Media for culturing stem cells

ABSTRACT

Well-defined, xeno-free culture media which comprise a TGF-beta isoform or the chimera formed between IL6 and the soluble IL6 receptor (IL6RIL6), which are capable of maintaining stem cells, and particularly, human embryonic stem cells, in an undifferentiated state are provided. Also provided are cell cultures comprising the culture media and the stem cells and methods of expanding and deriving embryonic stem cells in such well-defined, xeno-free culture media. In addition, the present invention provides methods of differentiating ESCs or EBs formed therefrom for the generation of lineage specific cells.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/909,128, filed on Jun. 4, 2013, which is a division of U.S. patentapplication Ser. No. 11/991,077 filed on Feb. 27, 2008, now U.S. Pat.No. 8,476,070, which is a National Phase of PCT Patent Application No.PCT/IL2006/000998 having International Filing Date of Aug. 29, 2006,which claims the benefit of priority of U.S. Provisional PatentApplication No. 60/834,795 filed on Aug. 2, 2006 and 60/711,668 filed onAug. 29, 2005. The contents of the above Applications are allincorporated herein by reference.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 66893SequenceListing.txt, created on Jul. 10,2016, comprising 12,288 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to culture media, cell cultures andmethods of culturing stem cells such as under defined and xeno-freeculturing conditions.

Human Embryonic stem cells (hESCs) were traditionally cultured andderived using the conventional methods employed for mouse ESCs, i.e., inthe presence of a medium supplemented with fetal bovine serum (FBS) andfeeder-layers consisting of inactivated mouse embryonic fibroblasts(MEFs) (Thomson et al, 1998). However, for use in cell-based therapy,hESC cultures should be well-defined and xeno-free (i.e., devoid of anyanimal contaminant) in terms of culture components. In recent years,extensive investigation into improving the culture systems for hESCs hasyielded the following advances: the ability to grow cells in serum-freeconditions (Amit et al, 2000); maintenance of the cells in anundifferentiated state on a Matrigel™ matrix with 100% MEF-conditionedmedium (Xu et al, 2001); and the use of either human embryonicfibroblasts, adult Fallopian tube epithelium (Richards et al, 2002) orforeskin fibroblasts (Amit et al, 2003; Hovatta et al, 2003) as feederlayers. However, while the use of MEF-conditioned medium or a Matrigel™matrix (which contains components from animal cells) may expose thehESCs to animal pathogens, the batch-specific variations may affect thequality of the culture. On the other hand, although humanfeeder-layer-based culture systems are xeno-free, they require thesimultaneous growth of both feeder cell layers and hESCs, which limitsthe potential of large-scale culturing of hESCs. Moreover, culturesystems based on feeder cells or conditioned medium are not well-definedand thus cannot be accurately repeated due to differences between thevarious lines of feeder-cells.

To overcome such limitations, attempts have been made to culture hESCsin feeder-layer-free culture systems devoid of conditioned medium. XuC., et al. (Stem Cells, 2005, 23:315-23) developed a culture systembased on a Matrigel™ matrix and a medium supplemented with serumReplacement™ (SR), basic fibroblast growth factor (bFGF), with orwithout the addition of the Flt-3 ligand to the culture medium. However,under these conditions, the background differentiation of the ESCs was20 or 28%, respectively, which is higher than observed for hESCs whencultured on MEFs. Another culturing system based a Matrigel™ matrix anda medium supplemented with bFGF and Noggin, an antagonist of bonemorphogenetic proteins (BMPs), resulted in a background differentiationof 10% (Xu R H., et al., 2005, Nat Methods. 2: 185-190). However, sinceboth of these systems rely on Matrigel™ as a culturing matrix, their usefor cell-based therapy is limited. To avoid animal contaminants, thepresent inventors have previously developed a culture system based on afibronectin matrix and a medium supplemented with 20% SR, transforminggrowth factor β1 (TGFβ₁) and bFGF (Amit et al, 2004). Under theseconditions, the cells maintained hESC features for more than 32passages. A further step towards defined culture conditions for hESCsculture was recently achieved by Ludwig and colleague (Ludwig et al,2006) when using a matrix consisted of the combination of human collagenIV, fibronectin, laminin and vitronectin and a medium supplemented withhuman serum albumin, bFGF and TGFβ₁. Such conditions enabled thederivation and culturing of hESCs under defined and feeder layer-freeculture conditions. However, hESCs cultured in these conditionsexhibited chromosomal instability of the cells following extendedperiods in culture. For example, one of the isolated hESC lines wasreported to harbor a karyotype of 47,XXY after 4 months of continuousculturing and a second line, which was initially normal, converted totrisomy 12 between 4 and 7 months of culturing. Thus, improvements ofthe feeder-free, xeno-free culturing systems of hESCs are highly needed.

Recent studies discussed the possible involvement of severalintracellular transduction pathways in hESC renewal and maintenance of“stemness” identity, but the mechanism underlining hESC self-maintenanceis still unrevealed. Sato and colleagues (Sato et al, 2004) suggestedthat the Wnt pathway is involved in hESC self-renewal. A laterpublication by the same group indicates that the TGFβ pathway plays acrucial role in cell-fate determination and holds interconnections withthe Wnt signaling pathway in maintaining hESC features (James, D., etal., 2005). These results are consistent with the feeder layer-freeculture method suggested by the present inventors (Amit et al, 2004),which is based on the addition of TGFβ₁, bFGF and/or LIF to a culturemedium which includes serum or serum replacement. In addition, themechanism by which bFGF involves in hESC′ self-maintenance has yet to beproven. Another candidate for the role of maintaining hESC properties isNoggin—an inhibitor of the BMPs signaling pathway (Xu R H., et al,2005). However, to date, not Noggin itself or any Noggin analog werefound in MEF-conditioned media.

Mouse ESCs can be continuously cultured without feeder layers providedthat leukemia inhibitory factor (LIF) is added to the culture medium.However, accumulating data regarding hESCs suggest that LIF has noeffect on preventing hESC differentiation (Thomson et al, 1998; Reubinofet al, 2000). In addition, activation of key proteins of the LIFcellular pathway, such as signal transducer and activator oftranscription 3 (STAT3) was found to be weak or absent in hESCs (Daheronet al, 2004; Humphrey et al, 2004; Sato et al, 2004). The gp130receptor, which is activated by ligands such as LIF, interleukin 6(IL-6) and a chimera made of IL-6 and its soluble IL6 receptor (theIL6RIL6 chimera; Chebath et al, 1997), was shown to positively affectthe mouse ESCs self-maintenance via STAT3 (Williams et al, 1988; Niwa etal, 1998; Smith et al, 1988). In hematopoietic stem cells, the IL6RIL6chimera exhibited a much higher affinity for human gp130 and was foundto be more potent in increasing proliferation of progenitor cells thanthe mixture of IL-6 and the soluble IL6 receptor (Kollet et al, 1999).On the other hand, the IL6RIL6 chimera induced differentiation ofESC-derived oligodendrocyte precursors (Zhang P L., et al., 2006, Mol.Cell Neurosci. 31: 387-398). In a recent study, Nichols et al., (1994)demonstrated that the IL6RIL6 chimera is capable of supporting mouse ESCculturing and derivation. On the other hand, Daheron et al. (2004)showed that although the LIFRβ and the signaling subunit gp130 areexpressed in hESCs and that human LIF can induce STATS phosphorylationand nuclear translocation in hESCs, human LIF is unable to maintain thepluripotent state of hESCs. In addition, Humphrey et al. (2004) foundthat hESCs rapidly differentiate when cultured in a medium containingmembers of the IL-6 family of cytokines such as LIF, IL-6 or a complexof the soluble IL-6 receptor and IL-6 (the “hyper-IL-6”) and concludedthat maintenance of pluropotency in human ESCs is STAT independent.Thus, it is currently accepted that in contrast to mouse ESCs which canbe maintained in the undifferentiated state in the presence ofactivators of the gp130 receptor, culturing of human ESCs in thepresence of LIF, IL6 or the hyper-IL-6 results in differentiation of thehESCs.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a defined, xeno-free medium suitable formaintaining stable, undifferentiated and pluripotent hESCs devoid of theabove limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided aculture medium being serum-free, xeno-free, feeder-free and proteincarrier-free and capable of maintaining stem cells in anundifferentiated state.

According to another aspect of the present invention there is provided acell culture comprising a stem cell and a culture medium, said culturemedium being serum-free, xeno-free, feeder-free and protein carrier-freeand capable of maintaining said stem cells in an undifferentiated state.

According to yet another aspect of the present invention there isprovided a method of expanding and maintaining stem cells in anundifferentiated state, the method comprising culturing the stem cellsin a culture medium being serum-free, xeno-free, feeder-free and proteincarrier-free and capable of maintaining the stem cells in anundifferentiated state, thereby expanding and maintaining the stem cellsin the undifferentiated state.

According to still another aspect of the present invention there isprovided a method of deriving an embryonic stem cell line, the methodcomprising: (a) obtaining an embryonic stem cell from a pre-implantationstage blastocyst, post-implantation stage blastocyst and/or a genitaltissue of a fetus; and (b) culturing said embryonic stem cell in aculture medium being serum-free, xeno-free, feeder-free and proteincarrier-free and capable of maintaining said embryonic stem cell in anundifferentiated state; thereby deriving the embryonic stem cell line.

According to an additional aspect of the present invention there isprovided a method of generating lineage-specific cells from embryonicstem cells, the method comprising: (a) culturing the embryonic stemcells in a culture medium being serum-free, xeno-free, feeder-free andprotein carrier-free and capable of maintaining the embryonic stem cellsin an undifferentiated state, to thereby obtain expanded,undifferentiated embryonic stem cells; (b) subjecting said expanded,undifferentiated embryonic stem cells to culturing conditions suitablefor differentiating and/or expanding lineage specific cells; therebygenerating the lineage-specific cells from the embryonic stem cells.

According to yet an additional aspect of the present invention there isprovided a method of generating embryoid bodies from embryonic stemcells, the method comprising: (a) culturing the embryonic stem cells ina culture medium being serum-free, xeno-free, feeder-free and proteincarrier-free and capable of maintaining the embryonic stem cells in anundifferentiated state, to thereby obtain expanded, undifferentiatedembryonic stem cells; and (b) subjecting said expanded, undifferentiatedembryonic stem cells to culturing conditions suitable fordifferentiating said embryonic stem cells to embryoid bodies; therebygenerating the embryoid bodies from the embryonic stem cells.

According to still an additional aspect of the present invention thereis provided a method of generating lineage-specific cells from embryonicstem cells, the method comprising: (a) culturing the embryonic stemcells in a culture medium being serum-free, xeno-free, feeder-free andprotein carrier-free and capable of maintaining the embryonic stem cellsin an undifferentiated state, to thereby obtain expanded,undifferentiated embryonic stem cells; (b) subjecting said expanded,undifferentiated embryonic stem cells to culturing conditions suitablefor differentiating said expanded, undifferentiated embryonic stem cellsto embryoid bodies; and (c) subjecting cells of said embryoid bodies toculturing conditions suitable for differentiating and/or expandinglineage specific cells; thereby generating the lineage-specific cellsfrom the embryonic stem cells.

According to a further aspect of the present invention there is provideda culture medium comprising a TGFβ isoform and being devoid of serum,serum replacement and protein carrier, wherein the culture medium iscapable of maintaining stem cells in an undifferentiated state.

According to yet a further aspect of the present invention there isprovided a cell culture comprising a stem cell and a culture medium,said culture medium comprising a TGFβ isoform and being devoid of serum,serum replacement and protein carrier, wherein said culture medium iscapable of maintaining said stem cell in an undifferentiated state.

According to still a further aspect of the present invention there isprovided a method of expanding and maintaining stem cells in anundifferentiated state, the method comprising culturing the stem cellsin a culture medium which comprises a TGFβ isoform and being devoid ofserum, serum replacement and protein carrier, said culture medium iscapable of maintaining the stem cells in an undifferentiated state,thereby expanding and maintaining the stem cells in the undifferentiatedstate.

According to still a further aspect of the present invention there isprovided a method of deriving an embryonic stem cell line, the methodcomprising: (a) obtaining an embryonic stem cell from a pre-implantationstage blastocyst, post-implantation stage blastocyst and/or a genitaltissue of a fetus; and (b) culturing said embryonic stem cell in aculture medium which comprises a TGFβ isoform and being devoid of serum,serum replacement and protein carrier, said culture medium is capable ofmaintaining the embryonic stem cells in an undifferentiated state,thereby deriving the embryonic stem cell line.

According to still a further aspect of the present invention there isprovided a method of generating lineage-specific cells from embryonicstem cells, the method comprising: (a) culturing the embryonic stemcells in a culture medium which comprises a TGFβ isoform and beingdevoid of serum, serum replacement and protein carrier, said culturemedium is capable of maintaining the embryonic stem cells in anundifferentiated state, to thereby obtain expanded, undifferentiatedembryonic stem cells; and (b) subjecting said expanded, undifferentiatedembryonic stem cells to culturing conditions suitable fordifferentiating and/or expanding lineage specific cells; therebygenerating the lineage-specific cells from the embryonic stem cells.

According to still a further aspect of the present invention there isprovided a method of generating embryoid bodies from embryonic stemcells, the method comprising: (a) culturing the embryonic stem cells ina culture medium which comprises a TGFβ isoform and being devoid ofserum, serum replacement and protein carrier, said culture medium iscapable of maintaining the embryonic stem cells in an undifferentiatedstate, to thereby obtain expanded, undifferentiated embryonic stemcells; and (b) subjecting said expanded, undifferentiated embryonic stemcells to culturing conditions suitable for differentiating saidembryonic stem cells to embryoid bodies; thereby generating the embryoidbodies from the embryonic stem cells.

According to still a further aspect of the present invention there isprovided a method of generating lineage-specific cells from embryonicstem cells, the method comprising: (a) culturing the embryonic stemcells in a culture medium which comprises a TGFβ isoform and beingdevoid of serum, serum replacement and protein carrier, said culturemedium is capable of maintaining the embryonic stem cells in anundifferentiated state, to thereby obtain expanded, undifferentiatedembryonic stem cells; (b) subjecting said expanded, undifferentiatedembryonic stem cells to culturing conditions suitable fordifferentiating said expanded, undifferentiated embryonic stem cells toembryoid bodies; and (c) subjecting cells of said embryoid bodies toculturing conditions suitable for differentiating and/or expandinglineage specific cells; thereby generating the lineage-specific cellsfrom the embryonic stem cells.

According to still a further aspect of the present invention there isprovided a culture medium comprising an IL6RIL6 chimera, wherein theculture medium is capable of maintaining human embryonic stem cells inan undifferentiated state.

According to still a further aspect of the present invention there isprovided a cell culture comprising a human embryonic stem cell and aculture medium, said culture medium comprising an IL6RIL6 chimera,wherein said culture medium is capable of maintaining said humanembryonic stem cell in an undifferentiated state.

According to still a further aspect of the present invention there isprovided a method of expanding and maintaining human embryonic stemcells in an undifferentiated state, the method comprising culturing thehuman embryonic stem cells in a culture medium which comprises anIL6RIL6 chimera, said culture medium is capable of maintaining the humanembryonic stem cells in an undifferentiated state, thereby expanding andmaintaining the embryonic stem cells in the undifferentiated state.

According to still a further aspect of the present invention there isprovided a method of deriving a human embryonic stem cell line, themethod comprising: (a) obtaining a human embryonic stem cell from apre-implantation stage blastocyst, post-implantation stage blastocystand/or a genital tissue of a fetus; and (b) culturing said humanembryonic stem cell in a culture medium which comprises an IL6RIL6chimera, said culture medium is capable of maintaining the humanembryonic stem cells in an undifferentiated state, thereby deriving theembryonic stem cell line.

According to still a further aspect of the present invention there isprovided a method of generating lineage-specific cells from embryonicstem cells, the method comprising: (a) culturing the embryonic stemcells in a culture medium which comprises an IL6RIL6 chimera, saidculture medium is capable of maintaining the embryonic stem cells in anundifferentiated state, to thereby obtain expanded, undifferentiatedembryonic stem cells; and (b) subjecting said expanded, undifferentiatedembryonic stem cells to culturing conditions suitable fordifferentiating and/or expanding lineage specific cells; therebygenerating the lineage-specific cells from the embryonic stem cells.

According to still a further aspect of the present invention there isprovided a method of generating embryoid bodies from embryonic stemcells, the method comprising: (a) culturing the embryonic stem cells ina culture medium which comprises an IL6RIL6 chimera, said culture mediumis capable of maintaining the embryonic stem cells in anundifferentiated state, to thereby obtain expanded, undifferentiatedembryonic stem cells; and (b) subjecting said expanded, undifferentiatedembryonic stem cells to culturing conditions suitable fordifferentiating said embryonic stem cells to embryoid bodies; therebygenerating the embryoid bodies from the embryonic stem cells.

According to still a further aspect of the present invention there isprovided a method of generating lineage-specific cells from embryonicstem cells, the method comprising: (a) culturing the embryonic stemcells in a culture medium which comprises an IL6RIL6 chimera, saidculture medium is capable of maintaining the embryonic stem cells in anundifferentiated state, to thereby obtain expanded, undifferentiatedembryonic stem cells; (b) subjecting said expanded, undifferentiatedembryonic stem cells to culturing conditions suitable fordifferentiating said expanded, undifferentiated embryonic stem cells toembryoid bodies; and (c) subjecting cells of said embryoid bodies toculturing conditions suitable for differentiating and/or expandinglineage specific cells; thereby generating the lineage-specific cellsfrom the embryonic stem cells.

According to further features in preferred embodiments of the inventiondescribed below, the embryonic stem cells are human embryonic stemcells.

According to still further features in the described preferredembodiments the culture medium comprising a TGFβ isoform.

According to still further features in the described preferredembodiments the culture medium being serum replacement-free.

According to still further features in the described preferredembodiments the culture medium comprising IL6RIL6 chimera.

According to still further features in the described preferredembodiments the culture medium comprising IL6RIL6 chimera and whereassaid stem cells are human embryonic stem cells.

According to still further features in the described preferredembodiments the culture medium comprising IL6RIL6 chimera and whereassaid embryonic stem cells are human embryonic stem cells.

According to still further features in the described preferredembodiments the stem cells are embryonic stem cells.

According to still further features in the described preferredembodiments the culture medium is capable of expanding said stem cellsin an undifferentiated state.

According to still further features in the described preferredembodiments the protein carrier is albumin.

According to still further features in the described preferredembodiments culturing is effected in suspension.

According to still further features in the described preferredembodiments the suspension is devoid of substrate adherence.

According to still further features in the described preferredembodiments culturing is effected on a feeder-layer free matrix.

According to still further features in the described preferredembodiments the feeder-layer free matrix is a fibronectin matrix.

According to still further features in the described preferredembodiments the culturing is effected on feeder cells.

According to still further features in the described preferredembodiments the culture medium is xeno-free.

According to still further features in the described preferredembodiments culturing is effected in xeno-free culturing conditions.

According to still further features in the described preferredembodiments the TGFβ isoform is a TGFβ isoform 1 (TGFβ₁).

According to still further features in the described preferredembodiments the TGFβ isoform is a TGFβ isoform 3 (TGFβ₃).

According to still further features in the described preferredembodiments the TGFβ₁ is provided at a concentration of at least 0.06ng/ml.

According to still further features in the described preferredembodiments the TGFβ₁ is provided at a concentration of 0.12 ng/ml.

According to still further features in the described preferredembodiments the TGFβ₃ is provided at a concentration of at least 0.5ng/ml.

According to still further features in the described preferredembodiments the TGFβ₃ is provided at a concentration of 2 ng/ml.

According to still further features in the described preferredembodiments the culture medium comprises basic fibroblast growth factor(bFGF).

According to still further features in the described preferredembodiments the bFGF is provided at a concentration of at least 2 ng/ml.

According to still further features in the described preferredembodiments the bFGF is provided at a concentration of at least 4 ng/ml.

According to still further features in the described preferredembodiments the IL6RIL6 chimera is provided at a concentration of atleast 25 ng/ml.

According to still further features in the described preferredembodiments the culture medium comprises serum or serum replacement.

According to still further features in the described preferredembodiments the culture medium is devoid of serum or serum replacement.

According to still further features in the described preferredembodiments the serum or serum replacement is provided at aconcentration of at least 10%.

According to still further features in the described preferredembodiments the method further comprising isolating lineage specificcells following step (b).

According to still further features in the described preferredembodiments isolating lineage specific cells is effected by a mechanicalseparation of cells, tissues and/or tissue-like structures containedwithin said embryoid bodies.

According to still further features in the described preferredembodiments isolating lineage specific cells is effected by subjectingsaid embryoid bodies to differentiation factors to thereby inducedifferentiation of said embryoid bodies into lineage specificdifferentiated cells.

According to still further features in the described preferredembodiments the embryonic stem cell is a human embryonic stem cell.

According to still further features in the described preferredembodiments the embryonic stem cell is a primate embryonic stem cell.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a well-defined, xeno-freeculture media which comprise a TGFβ isoform or the IL6RIL6 chimera,which are capable of maintaining stem cells in an undifferentiatedstate.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings executed in color. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of the preferred embodiments of the present invention only,and are presented in the cause of providing what is believed to be themost useful and readily understood description of the principles andconceptual aspects of the invention. In this regard, no attempt is madeto show structural details of the invention in more detail than isnecessary for a fundamental understanding of the invention, thedescription taken with the drawings making apparent to those skilled inthe art how the several forms of the invention may be embodied inpractice.

In the drawings:

FIGS. 1a-1f are photomicrographs depicting examples of the morphology ofundifferentiated ESC colonies and ESC single cells grown in variousfeeder-free culture systems. FIGS. 1a-1b —Undifferentiated I-3 colonycultured on a fibronectin-based feeder layer free culture system for 12passages in the presence of a culture medium supplemented with 100 ng/mlof the IL6RIL6 chimera. Note the “auto-feeders” formed at the peripheryof the colony (FIG. 1a , arrow). FIG. 1c —Undifferentiated I-3 colonycultured on a mouse laminin-based feeder layer free culture system for 7passages in the presence of a culture medium supplemented with 100 ng/mlof the IL6RIL6 chimera. Note the absence of “auto-feeders”. FIGS. 1d-1f—undifferentiated I-4 colonies cultured on a fibronectin-based feederfree culture system for 28 passages in the presence of a culture mediumsupplemented with 100 ng/ml of the IL6RIL6 chimera. Size bar for FIGS.1a, 1c, 1d, 1e,1f —20 μM; Size bar for FIG. 1b —50 μM.

FIG. 2 depicts a normal karyotype of an exemplary hESC. The karyotypewas examined after 7 passages of culturing in the presence of a culturemedium containing 300 ng/ml of the IL6RIL6 chimera on a fibronectinfeeder-free culture system. Note the presence of a normal 46,XXkaryotype. Normal karyotype was also detected when the hESCs werecultured for 7 passages in the presence of a culture medium containing100 ng/ml of the IL6RIL6 chimera on a fibronectin feeder-free culturesystem (not shown). Repeated test at passage 23 was found to be normal(not shown). 40 metaphases were examined from each sample.

FIGS. 3a-3c are photomicrographs depicting immunofluorescence stainingof undifferentiated colonies stained with surface markers specific tothe hESC undifferentiated stage. Undifferentiated I-4 hESCs werecultured on a fibronectin feeder-free culture system for 27 passages inthe presence of 100 ng/ml of the IL6RIL6 chimera and were subjected toimmunostatining with Tra-1-60 (FIG. 3a ), Oct 4 (FIG. 3b ) or SSEA-4(FIG. 3c ). Size bar=20 μM.

FIGS. 4a-4l are RT-PCR analyses depicting the expression ofrepresentative genes of the undifferentiated stage and of the threeembryonic germ layers in hESCs grown on human fibronectin feeder freeculture system or in embryoid bodies (EBs) derived therefrom. Lane1—Cell line I-3 cultured for 12 passages in CM100 (100 ng/ml of theIL6RIL6 chimera). Lane 2—Cell line I-3 cultured for 12 passages in 300ng/ml of the IL6RIL6 chimera. Lane 3—10-day-old EBs derived from I-3cells which were cultured for 10 passages in CM100. Lane 4—EBs derivedfrom I-3 cells which were cultured for 10 passages in 300 ng/ml of theIL6RIL6 chimera. FIG. 4a —Oct4; FIG. 4b —Sox2; FIG. 4c —Rex1; FIG. 4d—Cx43; FIG. 4e —FGF4; FIG. 4f —Albumin; FIG. 4g —Glucagon; FIG. 4h—β-Globulin; FIG. 4i —Cardiac actin; FIG. 4j —Flk1; FIG. 4k —AC133; FIG.4l —NFH.

FIGS. 5a-5d are photomicrographs depicting the morphology ofundifferentiated hES colonies and hES single cells cultured in variousculture systems in the presence of the TGFβ-containing culture media.FIG. 5a —I4 hESCs cultured for 28 passages on a Matrigel™ matrix withthe D1 medium; FIG. 5b —I4 hESCs cultured for 9 passages on MEFs withthe HA16 medium; FIG. 5c —I4 hESCs cultured for 20 passages on foreskinsfibroblasts with the D2 medium; FIG. 5d —I4 hESCs cultured for 11passages on a human fibronectin matrix with the D2 medium. Note theundifferentiated morphology after prolonged culturing with the uniqueTGFβ-containing media types. Magnifications are ×15 for FIGS. 5a -5 d.

FIGS. 6a-6c are photomicrographs depicting undifferentiated coloniesstained with surface markers specific to the hESC undifferentiatedstage. I4 hESCs cultured for 36 passages on a Matrigel™ matrix with themedium D1 and stained with TRA-1-60 (FIG. 6a ), SSEA4 (FIG. 6b ) andTRA-1-81 (FIG. 6c ); Magnifications are ×20 for FIGS. 6a -6 c.

FIGS. 7a-7b are photomicrographs depicting the derivation of a new hESCline under xeno-free conditions on foreskin fibroblasts using the HA16medium. FIG. 7a —the cultured embryo at first passage (p-1), arrowpoints at the inner cell mass (ICM); FIG. 7b —the isolated ICM atpassage 2 (p-2). Magnifications are ×20 for FIGS. 7a -b.

FIGS. 8a-8c are photomicrograph depicting immunostaining of hESCscultured for 3 passages in suspension in the presence of the D2 medium.Shown are immunostaining analyses of Oct4 (FIG. 8a ), TRA-1-60 (FIG. 8b) and TRA-1-81 (FIG. 8c ); Magnifications are ×63 for FIGS. 8a -8 c.

FIGS. 9a-9g are photomicrographs depicting histological sections andmorphology of suspended hESCs culture. FIG. 9a -Histology of a hESCclump (I4 hESC line) cultured for 3 passages in suspension in thepresence of the D1 medium and stained with H&E. Note that the hESC clumpis homogeneous, containing small cells with large nuclei typical forhESCs morphology. FIGS. 9b-9c —I4 hESCs were cultured for 3 passages insuspension in the presence of the D2 medium and were then re-cultured onMEFs. Shown is the morphology of the colonies after re-culturing on MEFsusing an inverted microscope. Note the typical undifferentiatedmorphology of the hESCs. FIGS. 9d-9e —I4 hESCs were cultured for 16passages in suspension in the presence of the CM100F medium and werethen re-cultured on MEFs. Shown is the morphology of colonies afterre-culturing on MEFs. Note the typical undifferentiated morphology ofthe hESCs. FIGS. 9f-9g —I4 hESCs were cultured for 7 passages insuspension in the presence of the HA19 medium (FIG. 9f ) or for 10passages in the presence of the CM100F medium (FIG. 9g ); Magnificationsare ×20 for FIG. 9a and ×15 for FIGS. 9b-9g . and

FIGS. 10a-10d are RT-PCR analyses depicting the expression ofrepresentative genes of the undifferentiated state of hESCs cultured insuspension in the presence of the HACM100, CM100F or the HA19 medium.Lane 1—I-4 hESCs cultured for 1 passage in suspension in the presence ofthe HACM100 medium (serum or serum replacement-free, IL6RIL6-containingmedium). Lane 2—I-4 hESCs cultured for 1 passage in suspension in thepresence of the CM100F medium (IL6RIL6 and serum replacement-containingmedium). Lane 3—I-4 hESCs cultured for 7 passages in suspension in thepresence of the HA19 medium (serum or serum replacement-free, proteincarrier-free, TGFβ₃-containing medium). Lane 4—I-4 hESCs cultured for 2passages in suspension in the presence of the HA19 medium and thenre-cultured on MEFs for additional 6 passages. FIG. 10a —Oct4; FIG. 10b—Rex1; FIG. 10c —Sox2; FIG. 10d —Nanog; RT mix were tested and foundnegative for all tested genes. All samples were tested for β-actin andwere found evenly positive.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of well-defined, xeno-free culture media whichcomprise a TGFβ isoform or the IL6RIL6 chimera, which are capable ofmaintaining stem cells in an undifferentiated state. In addition, thepresent invention is of cell cultures comprising the culture media andthe stem cells and of methods of expanding and deriving embryonic stemcells in such well-defined, xeno-free culture media. Moreover, thepresent invention is of methods of differentiating ESCs or EBs formedtherefrom for the generation of lineage specific cells. Specifically,the present invention can be used to generate highly reproducible,xeno-free cultures of hESCs which can be used for both cell-basedtherapy, pharmaceutical screening, identification of drug targets andcell-based compound delivery.

The principles and operation of the culture medium, cell culture andmethods according to the present invention may be better understood withreference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Human embryonic stem cells (hESCs) are proliferative, undifferentiated,stem cells capable of differentiating into cells of all three embryonicgerm layers. As such, hESCs are employed as a research model for earlyhuman development and hold promise for various applications includingcell-based therapy, pharmaceutical screening, identification of drugtargets and cell-based compound delivery which require almost indefiniteamounts of proliferating, yet pluripotent hESCs. For use in humantherapy and for the production of large amounts of hESCs, hESCs need tobe cultured in complete animal-free (xeno-free), well-defined culturesystems, where exposure to animal (e.g., retroviruses) or humanpathogens and batch-dependent variations are avoided.

Attempts to achieve defined and/or xeno-free hESCs culture systemsinclude the development of a culture system which is based on afibronectin matrix and a medium supplemented with 20% SR, transforminggrowth factor β₁ (TGFβ₁) and bFGF [Amit et al, 2004] or the culturesystem based on a matrix consisted of the combination of human collagenIV, fibronectin, laminin and vitronectin and a medium supplemented withhuman serum albumin, bFGF and TGFβ₁ (Ludwig et al, 2006). However, amajor drawback of the latter culture system is the chromosomalinstability of the cells associated with extended periods in culture.Thus, improvements of the feeder-free, xeno-free culturing systems ofhESCs are highly desirable.

One attractive approach of achieving a defined, xeno-free culture systemis using a culture medium which is based on isolated, recombinantfactors. For example, mouse ESCs can be continuously cultured withoutfeeder layers provided that leukemia inhibitory factor (LIF) is added tothe culture medium. However, accumulating data regarding hESCs suggestthat LIF has no effect on preventing hESC differentiation [Thomson etal, 1998; Reubinof et al, 2000]. A recombinant polypeptide, whichincludes the IL-6 and the soluble IL-6 receptor (the IL6RIL6 chimera,Chebath J, et al., 1997), was shown to support mouse ESC culturing andderivation (Nichols et al., 1994). However, a complex of the solublehIL-6R and hIL-(“hyper IL6”) failed to maintain human ESCs in theundifferentiated state when provided with a DMEM/serum replacement(DSR)/FGF2 medium on a laminin matrix (Humphrey et al, 2004). Thus, thecommon knowledge is that in contrast to mouse ESCs which can bemaintained in the undifferentiated state in the presence of activatorsof the gp130 receptor, culturing of human ESCs in the presence of LIF,IL6 or the “hyper IL6” results in differentiation of the hESCs.

While reducing the present invention to practice, the present inventorshave uncovered through laborious experimentations, a well-defined,xeno-free, serum or serum replacement-free and protein carrier-freeculture medium which comprises a TGFβ isoform and which can be used formaintaining hESCs in a pluripotent and undifferentiated state. As isshown in FIGS. 5a-5d and 6a-6c and is described in Example 2 of theExamples section which follows, hESCs cultured on a human fibronectinmatrix in the presence of a well-defined TGFβ-containing culture mediumwhich is devoid of serum, serum replacement and a protein carrier (e.g.,albumin) exhibited the morphology of undifferentiated ESC colonies andESC single cells (FIGS. 5a-5d ) and expressed typical hESC surfacemarkers specific to the undifferentiated state (FIGS. 6a-6c ). Inaddition, the present inventors have uncovered that the newTGFβ-containing culture medium is suitable for the successful derivationof new hESC lines (FIGS. 7a-7b , Example 3 of the Examples section whichfollows) on a complete xeno-free culture system.

Thus, according to one aspect of the present invention, there isprovided a culture medium. The culture medium comprises a TGFβ isoformand being devoid of serum, serum replacement and protein carrier,wherein the culture medium is capable of maintaining stem cells in anundifferentiated state.

As used herein the phrase “culture medium” refers to a solid or a liquidsubstance used to support the growth of stem cells and maintain them inan undifferentiated state. Preferably, the phrase “culture medium” asused herein refers to a liquid substance capable of maintaining the stemcells in an undifferentiated state. The culture medium used by thepresent invention can be a water-based medium which includes acombination of substances such as salts, nutrients, minerals, vitamins,amino acids, nucleic acids, proteins such as cytokines, growth factorsand hormones, all of which are needed for cell proliferation and arecapable of maintaining the stem cells in an undifferentiated state. Forexample, a culture medium according to this aspect of the presentinvention can be a synthetic tissue culture medium such as Ko-DMEM(Gibco-Invitrogen Corporation products, Grand Island, N.Y., USA),DMEM/F12 (Biological Industries, Biet Haemek, Israel), Mab ADCB medium(HyClone, Utah, USA) or DMEM/F12 (Biological Industries, Biet Haemek,Israel) supplemented with the necessary additives as is furtherdescribed hereinunder. Preferably, all ingredients included in theculture medium of the present invention are substantially pure, with atissue culture grade.

As used herein the phrase “stem cells” refers to cells which are capableof differentiating into other cell types or remaining in anundifferentiated state. Preferably, the phrase “stem cells” encompassesembryonic stem cells (ESCs), adult stem cells and hematopoietic stemcells.

The phrase “embryonic stem cells” refers to embryonic cells which arecapable of differentiating into cells of all three embryonic germ layers(i.e., endoderm, ectoderm and mesoderm), or remaining in anundifferentiated state. The phrase “embryonic stem cells” may comprisecells which are obtained from the embryonic tissue formed aftergestation (e.g., blastocyst) before implantation (i.e., apre-implantation blastocyst), extended blastocyst cells (EBCs) which areobtained from a post-implantation/pre-gastrulation stage blastocyst (seeWO2006/040763 to the present inventors] and embryonic germ (EG) cellswhich are obtained from the genital tissue of a fetus any time duringgestation, preferably before 10 weeks of gestation. Adult stem cells(also called “tissue stem cells”) include stem cells derived from anyadult or fetal tissue such as adipose tissue, skin, kidney, liver,prostate, pancreas, intestine and bone marrow. Hematopoietic stem cells,which may also referred to as adult tissue stem cells, include stemcells obtained from blood or bone marrow tissue of an individual at anyage or from cord blood of a newborn individual. Preferred stem cellsaccording to this aspect of the present invention are embryonic stemcells, preferably of a human or primate (e.g., monkey) origin.

The embryonic stem cells of the present invention can be obtained usingwell-known cell-culture methods. For example, human embryonic stem cellscan be isolated from human blastocysts. Human blastocysts are typicallyobtained from human in vivo preimplantation embryos or from in vitrofertilized (IVF) embryos. Alternatively, a single cell human embryo canbe expanded to the blastocyst stage. For the isolation of human ES cellsthe zona pellucida is removed from the blastocyst and the inner cellmass (ICM) is isolated by immunosurgery, in which the trophectodermcells are lysed and removed from the intact ICM by gentle pipetting. TheICM is then plated in a tissue culture flask containing the appropriatemedium which enables its outgrowth. Following 9 to 15 days, the ICMderived outgrowth is dissociated into clumps either by a mechanicaldissociation or by an enzymatic degradation and the cells are thenre-plated on a fresh tissue culture medium. Colonies demonstratingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and re-plated. Resulting ES cellsare then routinely split every 4-7 days. For further details on methodsof preparation human ES cells see Thomson et al., [U.S. Pat. No.5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998;Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can alsobe used with this aspect of the present invention. Human ES cells can bepurchased from the NIH human embryonic stem cells registry(http://escr.nih.gov). Non-limiting examples of commercially availableembryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92,CY10, TE03 and TE32.

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of atleast nine days post fertilization at a stage prior to gastrulation.Prior to culturing the blastocyst, the zona pellucida is digested [forexample by Tyrode's acidic solution (Sigma Aldrich, St Louis, Mo., USA)]so as to expose the inner cell mass. The blastocysts are then culturedas whole embryos for at least nine and no more than fourteen days postfertilization (i.e., prior to the gastrulation event) in vitro usingstandard embryonic stem cell culturing methods.

EG cells are prepared from the primordial germ cells obtained fromfetuses of about 8-11 weeks of gestation (in the case of a human fetus)using laboratory techniques known to anyone skilled in the arts. Thegenital ridges are dissociated and cut into small chunks which arethereafter disaggregated into cells by mechanical dissociation. The EGcells are then grown in tissue culture flasks with the appropriatemedium. The cells are cultured with daily replacement of medium until acell morphology consistent with EG cells is observed, typically after7-30 days or 1-4 passages. For additional details on methods ofpreparation human EG cells see Shamblott et al., [Proc. Natl. Acad. Sci.USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.

Adult tissue stem cells can be isolated using various methods known inthe art such as those disclosed by Alison, M. R. [J Pathol. 2003 200(5):547-50], Cal, J. et al., [Blood Cells Mol Dis. 2003 31(1): 18-27],Collins, A. T. et al., [J Cell Sci. 2001; 114(Pt 21): 3865-72], Potten,C. S. and Morris, R. J. [Epithelial stem cells in vivo. 1988. J. CellSci. Suppl. 10, 45-62], Dominici, M et al., [J. Biol. Regul. Homeost.Agents. 2001, 15: 28-37], Caplan and Haynesworth [U.S. Pat. No.5,486,359] Jones E. A. et al., [Arthritis Rheum. 2002, 46(12): 3349-60].Fetal stem cells can be isolated using various methods known in the artsuch as those disclosed by Eventov-Friedman S, et al., PLoS Med. 2006,3: e215; Eventov-Friedman S, et al., Proc Natl Acad Sci USA. 2005, 102:2928-33; Dekel B, et al., 2003, Nat Med. 9: 53-60; and Dekel B, et al.,2002, J. Am. Soc. Nephrol. 13: 977-90. Hematopoietic stem cells can beisolated using various methods known in the arts such as those disclosedby “Handbook of Stem Cells” edit by Robert Lanze, Elsevier AcademicPress, 2004, Chapter 54, pp 609-614, isolation and characterization ofhematopoietic stem cells, by Gerald J Spangrude and William B Stayton.

It will be appreciated that stem cells in an undifferentiated state areof a distinct morphology, which is clearly distinguishable by theskilled in the art from that of differentiated cells of embryo or adultorigin. Typically, undifferentiated stem cells have highnuclear/cytoplasmic ratios, prominent nucleoli and compact colonyformation with poorly discernable cell junctions. Additional features ofthe undifferentiated state of the stem cells are further describedhereinunder.

The culture medium according to this aspect of the present inventioncomprises a TGFβ isoform and being devoid of serum, serum replacementand protein carrier.

As used herein the phrase “TGFβ isoform” refers to any isoform of thetransforming growth factor beta (β) including TGFβ₁ (e.g., homo sapiensTGFβ₁, GenBank Accession No. NP_000651), TGFβ₂ (e.g., homo sapiensTGFβ₂, GenBank Accession No. NP_003229) and TGFβ₃ (e.g., homo sapiensTGFβ₃, GenBank Accession No. NP_003230) which function through the samereceptor signaling system in the control of proliferation,differentiation, and other functions in many cell types. TGFβ acts ininducing transformation and also acts as a negative autocrine growthfactor. According to preferred embodiments of the present invention theTGFβ isoform which is included in the culture medium of the presentinvention is TGFβ₁ or TGFβ₃. Such TGFβ isoforms can be obtained fromvarious commercial sources such as R&D Systems Minneapolis Minn., USA.

As is shown in Example 2 of the Examples section which follows, thepresent inventors have used various culture media which contain TGFβ₁(e.g., the D1 medium which contains 0.12 ng/ml TGFβ₁) or TGFβ₃ (e.g.,the D2 medium, the HA16 medium or the HA19 medium which contain 2 ng/mlTGFβ₃) to successfully culture hESCs and maintain them in theundifferentiated state.

Preferably, TGFβ₁ which is included in the culture medium of this aspectof the present invention is provided at a concentration of at least 0.06ng/ml, more preferably, at least 0.07 ng/ml, more preferably, at least0.08 ng/ml, more preferably, at least 0.09 ng/ml, more preferably, atleast 0.1 ng/ml, more preferably, at least 0.11 ng/ml, even morepreferably, at least 0.12 ng/ml.

Preferably, TGFβ₃ which is included in the culture medium of this aspectof the present invention is provided at a concentration of at least 0.5ng/ml, more preferably, at least 0.6 ng/ml, more preferably, at least0.8 ng/ml, more preferably, at least 0.9 ng/ml, more preferably, atleast 1 ng/ml, more preferably, at least 1.2 ng/ml, more preferably, atleast 1.4 ng/ml, more preferably, at least 1.6 ng/ml, more preferably,at least 1.8 ng/ml, even more preferably, at least 2 ng/ml.

Preferably, the TGFβ-containing culture medium of this aspect of thepresent invention further includes other growth factors such as basicfibroblast growth factor (bFGF). bFGF can be obtained from anycommercial supplier of tissue culture ingredients such as InvitrogenCorporation products, Grand Island N.Y., USA.

As is shown in Example 2 of the Examples section which follows, aDMEM/F12-based culture medium (e.g., HA16 or HA19 medium which includesTGFβ₃ and 4 ng/ml bFGF) or Mab ADCB medium-based culture medium (e.g.,D1 or D2 medium which includes TGFβ₁ or TGFβ₃, respectively, and 10ng/ml bFGF) were capable of maintaining hESCs in the undifferentiatedstate in culture. It should be mentioned that a Mab ADCB medium-basedculture medium which includes a TGFβ₁ or TGFβ₃ isoform and bFGF at aconcentration of 8 ng/ml was also capable of maintaining hESCs in theundifferentiated state for at least 5 passages (data not shown).

Preferably, the bFGF which is included in TGFβ-containing culture mediumof this aspect of the present invention is provided at a concentrationof at least 2 ng/ml, at least 3 ng, at least 4 ng/ml, at least 5 ng/ml,at least 6 ng/ml, at least 7 ng, at least 8 ng/ml, at least 9 ng/ml, atleast 10 ng/ml.

As mentioned, the culture medium of this aspect of the present inventionis devoid of a protein carrier (i.e., protein carrier-free). A proteincarrier refers to a protein which acts in the transfer of proteins ornutrients (e.g., minerals such as zinc) to the cells in the culture.Such protein carriers can be, for example, albumin (e.g., bovine serumalbumin), Albumax (lipid enriched albumin) or plasmanate (human plasmaisolated proteins). Since these carriers are derived from either humanor animal sources their use in hESCs cultures is limited bybatch-specific variations and/or exposure to pathogens. On the otherhand, the recombinant human albumin, which is substantially pure anddevoid of animal contaminants is highly expensive, thus not commonlyused in hESCs cultures. Thus, a culture medium which is devoid of aprotein carrier is highly advantageous since it enables a truly definedmedium that can be manufacture from recombinant or synthetic materials.

In addition, as mentioned hereinabove, the culture medium of this aspectof the present invention is also devoid of serum (i.e., serum-free) orserum replacement (i.e., serum replacement-free). It should be notedthat serum or serum replacement are added to most culture media whichare designed for culturing stem cells, and particularly, embryonic stemcells, in order to provide the cells with the optimal environment,similar to that present in vivo (i.e., within the organism from whichthe cells are derived, e.g., a blastocyst of an embryo or an adulttissue of a postnatal individual). However, while the use of serum whichis derived from either an animal source (e.g., bovine serum) or a humansource (human serum) is limited by the significant variations in serumcomponents between individuals and the risk of having xeno contaminants(in case of an animal serum is used), the use of the more definedcomposition such as the currently available serum Replacement™(Gibco-Invitrogen Corporation, Grand Island, N.Y. USA) may be limited bythe presence of Albumax (Bovine serum albumin enriched with lipids)which is from an animal source within the composition (InternationalPatent Publication No. WO 98/30679 to Price, P. J. et al).

Thus, a culture medium which comprises a TGFβ isoform as describedhereinabove and is devoid of serum, serum replacement and a proteincarrier is highly desirable for both cell-based therapy andpharmaceutical industry, e.g., for pharmaceutical screening,identification of drug targets and cell-based compound delivery.

Preferably, the culture medium of this aspect of the present inventionis capable of expanding the stem cells while maintaining them in theundifferentiated state. As used herein the term “expanding” refers toobtaining a plurality of cells from a single or a population of stemcells. Preferably, expanding embryonic stem cells refers also toincreasing the number of embryonic stem cells over the culturing period.It will be appreciated that the number of stem cells which can beobtained from a single stem cell depends on the proliferation capacityof the stem cell. The proliferation capacity of a stem cell can becalculated by the doubling time of the cell (i.e., the time needed for acell to undergo a mitotic division in the culture) and the period thestem cell culture can be maintained in the undifferentiated state (whichis equivalent to the number of passages multiplied by the days betweeneach passage).

For example, as described in Example 2 of the Examples section whichfollows, hESCs could be maintained in the undifferentiated state in thepresence of the D1 TGFβ-containing culture medium for at least 53passages. Given that each passage occurs every 4-7 days, the hESCs weremaintained for 265 days (i.e., 6360 hours). Given that the hESCsdoubling time was 36 hours, a single hESC cultured under theseconditions could be expanded to give rise to 2¹⁷⁶ (i.e., 9.57×10⁵²)hESCs.

Preferably the stem cells which are maintained and expanded in theculture medium of the present invention exhibit stable karyotype(chromosomal stability) while in culture. For example, hESCs cultured inthe presence of an IL6RIL6-containing medium (e.g., CM100) or aTGFβ-containing medium (e.g., D1, D2 or HA16) exhibited normal karyotypefollowing at least 23 or 15 passages, respectively.

While further reducing the present invention to practice, the presentinventors have uncovered that a culture medium which includes theIL6RIL6 chimera is also capable of maintaining human ESCs in theundifferentiated state. This is in sharp contrast to the teachings ofHumphrey R., et al., (2004) which failed to maintain hESCs in theundifferentiated state when using the “hyper IL6” complex and thusconcluded that maintenance of pluropotency in human ESCs is STATindependent. Thus, Humphrey R., et al., (2004) teaches away the presentinvention.

As is shown in FIGS. 1a-1f , 2, 3 a-3 c and 4 a-4 l and described inExample 1 of the Examples section which follows, a culture system basedon a fibronectin or laminin feeder layer-free matrix and a culturemedium which includes the IL6RIL6 chimera, serum replacement and bFGFwas capable of maintaining hESCs in the undifferentiated state for atleast 43 (on a fibronectin matrix) or 7 (on a laminin matrix) passageswhile preserving all hESCs characteristics and pluripotency. On theother hand, as is further shown in Example 1 of the Examples sectionwhich follows, a medium containing serum replacement, bFGF and theunconjugated chimera components, i.e., IL-6 (GenBank Accession No.CAG29292) and soluble IL-6 receptor (GenBank Accession No. AAH89410),failed to support hESC prolonged culture and resulted in differentiationof hESCs within 3-5 passages. In addition, hESCs cultured in a mediumcontaining the IL6RIL6 chimera and serum replacement, in the absence ofbFGF, exhibited low proliferation capacity and could not be maintainedin culture beyond 1-2 passages. Thus, these results demonstrate, for thefirst time, that hESCs can be cultured and maintained in theundifferentiated state in a feeder-layer free culture system in thepresence of a culture medium which comprises the IL6RIL6 chimera.

Thus, according to another aspect of the present invention there isprovided a culture medium which comprises an IL6RIL6 chimera, whereinthe culture medium is capable of maintaining human embryonic stem cellsin an undifferentiated state.

As used herein the term “IL6RIL6” refers to a chimeric polypeptide whichcomprises the soluble portion of interleukin-6 receptor (IL-6-R, e.g.,the human IL-6-R as set forth by GenBank Accession No. AAH89410) (e.g.,a portion of the soluble IL6 receptors as set forth by amino acids112-355 of GenBank Accession No. AAH89410) and the interleukin-6 (IL6)(e.g., human IL-6 as set forth by GenBank Accession No. CAG29292) or abiologically active fraction thereof (e.g., a receptor binding domain).Preferably, the IL6RIL6 chimera used by the method according to thisaspect of the present invention is capable of supporting theundifferentiated growth of human embryonic stem cells, while maintainingtheir pluripotent capacity. It will be appreciated that whenconstructing the IL6RIL6 chimera the two functional portions (i.e., theIL6 and its receptor) can be directly fused (e.g., attached ortranslationally fused, i.e., encoded by a single open reading frame) toeach other or conjugated (attached or translationally fused) via asuitable linker (e.g., a polypeptide linker). Preferably, the IL6RIL6chimeric polypeptide exhibits a similar amount and pattern ofglycosylation as the naturally occurring IL6 and IL6 receptor. Forexample, a suitable IL6RIL6 chimera is as set forth in SEQ ID NO:31 andin FIG. 11 of WO 99/02552 to Revel M., et al., which is fullyincorporated herein by reference.

It will be appreciated that any of the proteinaceous factors used in theculture medium of the present invention (e.g., the IL6RIL6 chimera,bFGF, TGFβ₁, TGFβ₃) can be recombinantly expressed or biochemicallysynthesized. In addition, naturally occurring proteinaceous factors suchas bFGF and TGFβ can be purified from biological samples (e.g., fromhuman serum, cell cultures) using methods well known in the art.

Biochemical synthesis of the proteinaceous factors of the presentinvention (e.g., the IL6RIL6 chimera) can be performed using standardsolid phase techniques. These methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensationand classical solution synthesis.

Recombinant expression of the proteinaceous factors of the presentinvention (e.g., the IL6RIL6 chimera) can be generated using recombinanttechniques such as described by Bitter et al., (1987) Methods inEnzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol.185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al.(1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680,Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol.Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp 421-463.Specifically, the IL6RIL6 chimera can be generated as described in PCTpublication WO 99/02552 to Revel M., et al. and Chebath J, et al., 1997,which are fully incorporated herein by reference.

For example, to generate the IL6RIL6 chimera, a polynucleotide sequenceencoding the IL6RIL6 chimera (e.g., the polypeptide set forth by SEQ IDNO:31) is preferably ligated into a nucleic acid construct suitable forexpression in a host cell [i.e., a cell in which the polynucleotideencoding the polypeptide-of-choice (e.g., the IL6RIL6 chimera) isexpressed]. Preferably, to generate an IL6RIL6 chimera with the amountand pattern of glycosylation as of the naturally occurring IL6 andIL6-R, the host cell employed is a eukaryotic host cell, more preferablya mammalian host cell such as human cell or CHO cell).

For expression in mammalian cells [e.g., CHO cells, human HEK 293 cells(ATCC CRL 1573)] a number of mammalian expression vectors can be used.Examples include, but are not limited to, pcDNA3, pcDNA3.1(+/−), pGL3,pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1,pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available fromInvitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSVand pBK-CMV which are available from Strategene, pTRES which isavailable from Clontech, and their derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

Various methods can be used to introduce the expression vector of thepresent invention into host cells. Such methods are generally describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory, New York (1989, 1992), in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995),Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4(6): 504-512, 1986] and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Transformed cells are cultured under effective conditions, which allowfor the expression of high amounts of the recombinant polypeptide (e.g.,the IL6RIL6 chimera). Following a predetermined time in culture,recovery of the recombinant polypeptide is effected. The phrase“recovery of the recombinant polypeptide” used herein refers tocollecting the whole fermentation medium containing the polypeptide andneed not imply additional steps of separation or purification.

Thus, polypeptides of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

The polypeptide of the present invention (e.g., the IL6RIL6 chimera) ispreferably retrieved in “substantially pure” form. As used herein, thephrase “substantially pure” refers to a purity that allows for theeffective use of the polypeptide of the present invention (e.g., theIL6RIL6 chimera) in maintaining the human embryonic stem cells in anundifferentiated state while in culture.

As shown in FIGS. 1-4 and described in Example 1 of the Examples sectionwhich follows, a medium supplemented with the 100 or 300 ng/ml of theIL6RIL6 chimera enabled the prolonged culturing of hESCs in theundifferentiated state for at least 43 passages, while preserving normalkaryotype and pluripotent capacity [as evidenced by the formation ofembryoid bodies (EBs) which express markers of all three embryonic germcells].

Preferably, the IL6RIL6 chimera which is included in the culture mediumof this aspect of the present invention is provided at a concentrationof at least 25 ng/ml, at least 50 ng/ml, at least 100 ng/ml, preferably,at least 200 ng/ml, preferably, at least 300 ng/ml. It should be notedthat the concentration of the IL6RIL6 chimera can vary depending on thepurity of the chimeric polypeptide following its synthesis orrecombinant expression and those of skills in the art are capable ofadjusting the optimal concentration depending on such purity.

Preferably, the IL6RIL6-containing culture medium of this aspect of thepresent invention includes at least 2 ng/ml bFGF, at least 3 ng/ml, atleast 4 ng/ml, at least 5 ng/ml, at least 6 ng/ml, at least 7 ng, atleast 8 ng/ml, at least 9 ng/ml, at least 10 ng/ml bFGF. For example, asshown in Example 1 of the Examples section which follows, a culturemedium which includes 4 ng/ml bFGF along with 100 ng/ml of the IL6RIL6chimera was capable of maintaining hESCs in an undifferentiated statefor at least 43 passages.

As mentioned, the IL6RIL6-containing culture medium described in Example1 of the Examples section which follows included serum replacement. Itshould be noted that such a culture medium can also include serum (e.g.,such as human serum) instead of serum replacement and yet maintain hESCsin culture in the undifferentiated state. Thus, serum or serumreplacement which are included in the IL6RIL-containing culture mediumof this aspect of the present invention can be provided at variousconcentrations, such as a concentration of at least 10%, e.g., aconcentration of at least 15%, at least 20%, at least 25% or at least30%.

Alternatively and currently more preferred, in order to achieve moredefined culture conditions of the hESCs, the IL6RIL6-containing culturemedium of this aspect of the present invention is preferably devoid ofserum or serum replacement. A non-limiting example of such a culturemedium can be the HACM100 culture medium described in Examples 2 and 4of the Examples section which follows.

Thus, any of the culture media described hereinabove, which are based onthe TGFβ isoform or the IL6RIL6 chimera present, for the first time, awell-defined, xeno-free culture medium which is highly suitable forculturing hESCs for applications such as cell-based therapy and for usein the pharmaceutical industry.

Thus, according to yet another aspect of the present invention there isprovided a culture medium which is devoid of serum (i.e., serum-free),xeno contaminant (i.e., xeno-free), feeder layers (i.e., feederlayer-free) and protein carrier (i.e., protein carrier-free) and yetcapable of maintaining stem cells in an undifferentiated state.

As mentioned hereinabove and described in Examples 1 and 2 of theExamples section which follows, the present inventors have uncoveredthat stem cells such as human embryonic stem cells can be expanded usingany of the culturing media described hereinabove and be maintained inthe undifferentiated state while in culture.

Thus, according to an additional aspect of the present invention thereis provided a cell culture which comprises the stem cell and any of theculture media described hereinabove, while the culture medium is capableof maintaining said stem cells in an undifferentiated state.

According to still an additional aspect of the present invention thereis provided a method of expanding and maintaining stem cells in anundifferentiated state. The method is effected by culturing the stemcells in any of the culture media described hereinabove, therebyexpanding and maintaining the stem cells in the undifferentiated state.

Culturing according to this aspect of the present invention is effectedby plating the stem cells onto a matrix in a cell density which promotescell survival and proliferation but limits differentiation. Typically, aplating density of between about 15,000 cells/cm² and about 3,000,000cells/cm² is used.

It will be appreciated that although single-cell suspensions of stemcells are usually seeded, small clusters may also be used. To this end,enzymatic digestion (such as with type IV collagenase) utilized forcluster disruption (see Examples 1 and 2 of the Examples section whichfollows) is terminated before stem cells become completely dispersed andthe cells are triturated with a pipette such that clumps (i.e., 10-200cells) are formed. However, measures are taken to avoid large clusterswhich may cause cell differentiation.

As mentioned before, stem cells can be cultured on feeder cells or onfeeder-layer free culturing systems using a matrix instead of a feedercell layer. As used herein, the term “matrix” refers to any substance towhich the stem cells can adhere and which therefore can substitute thecell attachment function of feeder cells. Such a matrix typicallycontains extracellular components to which the stem cells can attach andthus it provides a suitable culture substrate.

Particularly suitable for use with the present invention areextracellular matrix components derived from basement membrane orextracellular matrix components that form part of adhesion moleculereceptor-ligand couplings. Non-limiting examples of suitable matriceswhich can be used by the method of this aspect of the present inventioninclude Matrigel® (Becton Dickinson, USA), laminin, fibronectin,proteoglycan, entactin, heparan sulfate, and the like, alone or invarious combinations. In cases where complete animal-free culturingconditions are desired, the matrix is preferably derived from a humansource or synthesized using recombinant techniques. Such matricesinclude, for example, human-derived fibronectin, recombinantfibronectin, human-derived laminin, foreskin fibroblast matrix or asynthetic fibronectin matrix which can be obtained from Sigma, St.Louis, Mo., USA or can be produced using known recombinant DNAtechnology. Preferred matrices of the present invention are fibronectinderived matrices.

Thus, stem cells cultured using the teachings of the present inventioncan be expanded while maintaining in the undifferentiated state. Forexample, as described in Example 2 of the Examples section whichfollows, culturing of hESCs on a Matrigel™ in the presence of theTGFβ-containing culture medium (the D1 medium) for at least 53 passages(8-9 months) resulted in an expansion factor of 2¹⁸⁰ (i.e., 1.5×10⁵⁴)given that the hESC doubling time is 36 hours and that passaging occursevery 4-6 days.

Alternatively, as is shown in FIGS. 8a-8c, 9a-9g and 10a-10d and isdescribed in Example 4 of the Examples section which follows and in U.S.provisional application No. 60/834,795 to the present inventors (filedAug. 2, 2006), the present inventors have uncovered that human ESCs canbe expanded in a suspension culture devoid of substrate adherence and assuch can be maintained in the undifferentiated, pluripotent state.

As used herein the phrase “suspension culture” refers to a culture inwhich the embryonic stem cells are suspended in a medium rather thanadhering to a surface.

Thus, the culture of the present invention is “devoid of substrateadherence” in which the stem cells (e.g., ESCs) are capable of expandingwithout adherence to an external substrate such as components ofextracellular matrix, a glass microcarrier or beads.

Thus, hESCs cultured in a suspension culture devoid of substrateadherence can be expanded for at least 17 passages in the CM100F mediumand maintain their undifferentiated, pluripotent state (Example 4 anddata not shown).

When cultured according to the teachings of the present invention, stemcell growth is monitored to determine their differentiation state. Thedifferentiation state can be determined using various approachesincluding, for example, morphological evaluation (e.g., as shown inFIGS. 1a-1f, 5a-5d and 9a-9g ) and/or detection of the expressionpattern of typical markers of the undifferentiated state usingimmunological techniques such as flow cytometry for membrane-boundmarkers, immunohistochemistry or immunofluorescence for extracellularand intracellular markers and enzymatic immunoassay, for secretedmolecular markers. For example, immunofluorescence employed on hESCscultured according to the method of this aspect of the present inventionrevealed the expression of Oct4, stage-specific embryonic antigen (SSEA)4, the tumour-rejecting antigen (TRA)-1-60 and TRA-1-81 (FIGS. 3a-3c,6a-6c and 8a-8c ). Additionally, the level of transcripts of specificundifferentiation markers (e.g., Oct 4, Nanog, Sox2, Rex1, Cx43, FGF4)or differentiation markers (e.g., albumin, glucagons, α-cardiac actin,β-globulin, Flk1, AC133 and neurofilament) can be detected usingRNA-based techniques such as RT-PCR analysis (as shown in FIGS. 4a-4land 10a-10d ) and/or cDNA microarray analysis.

Determination of ES cell differentiation can also be effected viameasurements of alkaline phosphatase activity. Undifferentiated human EScells have alkaline phosphatase activity which can be detected by fixingthe cells with 4% paraformaldehyde and developing with the Vector Redsubstrate kit according to manufacturer's instructions (VectorLaboratories, Burlingame, Calif., USA).

As is further shown in FIGS. 7a-7b and described in Example 3 of theExamples section which follows, the present inventors were capable ofderiving a new line of human ESCs in complete xeno-free culturingconditions using the TGFβ-based culture medium.

Thus, according to yet an additional aspect of the present inventionthere is provided a method of deriving an embryonic stem cell line. Themethod is effected by: (a) obtaining an embryonic stem cell from apre-implantation stage blastocyst, post-implantation stage blastocystand/or a genital tissue of a fetus; and (b) culturing the embryonic stemcell in any of the culture media described hereinabove, thereby derivingthe embryonic stem cell line.

The term “deriving” as used herein refers to generating an embryonicstem cell line from at least one embryonic stem cell.

As used herein the phrase “embryonic stem cell line” refers to embryonicstem cells which are derived from a single or a group of embryonic stemcells of a single organism (e.g., a single human blastocyst), and whichare characterized by the ability to proliferate in culture whilemaintaining the undifferentiated state and the pluripotent capacity.

Obtaining an embryonic stem cell from a pre-implantation stageblastocyst, post-implantation stage blastocyst and/or a genital tissueof a fetus can be performed using methods known in the art, as describedhereinabove and in the “General Materials and Experimental Methods” ofthe Examples section which follows. Briefly, the zona pellucida isremoved from a 5-7 day-old blastocyst using Tyrode's acidic solution(Sigma, St Louis Mo., USA), the trophoblast layer is specificallyremoved either by immunosurgery or mechanically using 27 g needles andthe exposed ICM is either directly cultured in a suitable culture system(e.g., feeder layers, feeder-free matrix or a suspension culture) in thepresence of any of the culture media described hereinabove (e.g., theCM100F, HA16 or D2 medium) for 4-10 days (in case a preimplantationblastocyst is used) or subject to in vitro implantation by culturing theICM for 6-8 days (to obtain cells of a 13 day-old blastocyst in case apost-implantation/pre-gastrulation blastocyst is used) on feeder layersor a feeder-free culturing system which allow implantation of theblastocyst to the surface, following which the implanted cells areisolated and can be further cultured on feeder layers, feeder-freematrix or a suspension culture in the presence of any of the culturemedia described hereinabove (e.g., the CM100F, HA16 or D2 medium) asdescribed hereinunder. When using the genital tissue of a fetus, thegenital ridges are dissociated and cut into small chunks which arethereafter disaggregated into cells by mechanical dissociation. Thesingle cell EG cells are then cultured in any of the culture mediadescribed hereinabove for 4-10 days).

Once obtained the ESCs are further cultured in any of the culture mediadescribed hereinabove which allow expansion of the embryonic stem cellsin the undifferentiated state, essentially as described hereinabove.

Preferably, the cell culture of the present invention is characterizedby at least 40%, at least 50%, at least 60%, more preferably at least70%, more preferably at least 80%, most preferably at least 85% ofundifferentiated stem cells.

It will be appreciated that an established embryonic stem cell line canbe subject to freeze/thaw cycles without hampering the proliferativecapacity of the cells in the undifferentiated state while preservingtheir pluripotent capacity. For example, as is shown in the Examplessection which follows, using 15% SR and 10% DMSO, hESCs weresuccessfully frozen and thawed.

As is shown in FIGS. 4a-4l and is described in Examples 1, 2 and 4 ofthe Examples section which follows, hESCs which were expanded andmaintained in any of the culture media described hereinabove arepluripotent (i.e., capable of differentiating into all cell types of thethree embryonic germ layers, the ectoderm, the endoderm and themesoderm) as evidenced in vitro (by the formation of EBs) and in vivo(by the formation of teratomas). Thus, hESCs cultured according to theteachings of the present invention can be used as a source forgenerating differentiated, lineage-specific cells. Such cells can beobtained directly from the ESCs by subjecting the ESCs to variousdifferentiation signals (e.g., cytokines, hormones, growth factors) orindirectly, via the formation of embryoid bodies and the subsequentdifferentiation of cells of the EBs to lineage-specific cells.

Thus, according to yet an additional aspect of the present inventionthere is provided a method of generating embryoid bodies from embryonicstem cells. The method is effected by (a) culturing the embryonic stemcells in any of the culture media described hereinabove which is capableof maintaining the embryonic stem cells in an undifferentiated state, tothereby obtain expanded, undifferentiated embryonic stem cells; and (b)subjecting the expanded, undifferentiated embryonic stem cells toculturing conditions suitable for differentiating the embryonic stemcells to embryoid bodies; thereby generating the embryoid bodies fromthe embryonic stem cells.

As used herein the phrase “embryoid bodies” refers to morphologicalstructures comprised of a population of ESCs, extended blastocyst cells(EBCs) and/or embryonic germ cells (EGCs) which have undergonedifferentiation. EBs formation initiates following the removal ofdifferentiation blocking factors from ES cell cultures. In the firststep of EBs formation, ESCs proliferate into small masses of cells whichthen proceed with differentiation. In the first phase ofdifferentiation, following 1-4 days in culture for human ESCs, a layerof endodermal cells is formed on the outer layer of the small mass,resulting in “simple EBs”. In the second phase, following 3-20 dayspost-differentiation, “complex EBs” are formed. Complex EBs arecharacterized by extensive differentiation of ectodermal and mesodermalcells and derivative tissues.

Thus, the method of this aspect of the present invention involves theculturing of ESCs in any of the culture media described hereinabove inorder to obtain expanded, undifferentiated embryonic stem cells and thensubjecting the expanded, undifferentiated ESCs to culturing conditionssuitable for differentiating the ESCs to embryoid bodies. Such culturingconditions are substantially devoid of differentiation inhibitoryfactors which were employed during step (a), e.g., a TGFβ isoform or theIL6RIL6 chimera.

For EBs formation, the ESCs are removed from their feeder cell layers,feeder-free-culturing systems (e.g., the fibronectin or laminin matrix)or suspension cultures and are transferred to a suspension culture inthe presence of a culture medium containing serum or serum replacementand being devoid of differentiation-inhibitory factors, essentially asdescribed in Examples 1, 2 and 4 of the Examples section which follows.For example, a culture medium suitable for EBs formation may include abasic culture medium (e.g., Ko-DMEM or DMEM/F12) supplemented with 20%FBSd (HyClone, Utah, USA), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol,and 1% non-essential amino acid stock.

Monitoring the formation of EBs can be effected by morphologicalevaluations (e.g., histological staining), determination of expressionof differentiation-specific markers [e.g., using immunologicaltechniques or RNA-based analysis (e.g., RT-PCR, cDNA microarray) asshown in FIGS. 4a-4l ], essentially as described in the Examples sectionwhich follows.

Thus, as is shown in FIGS. 4f-4l , cells harvested from EBs according tothe method of this aspect of the present invention exhibited markers ofall three embryonic germ layers, such as albumin and glucagon (typicalof the embryonic endoderm), α-cardiac actin, β-globulin and Flk1(typical of the embryonic mesoderm), and AC133 and neurofilament (NFH)(typical of the embryonic ectoderm).

It will be appreciated that in order to obtain lineage-specific cellsfrom the EBs, cells of the EBs can be further subjected to culturingconditions suitable for lineage-specific cells.

Preferably, the method of this aspect of the present invention furtherincludes step (c) of subjecting cells of the embryoid bodies toculturing conditions suitable for differentiating and/or expandinglineage specific cells; thereby generating the lineage-specific cellsfrom the embryonic stem cells.

As used herein the phrase “culturing conditions suitable fordifferentiating and/or expanding lineage specific cells” refers to acombination of culture system, e.g., feeder cell layers, feeder-freematrix or a suspension culture and a culture medium which are suitablefor the differentiation and/or expansion of specific cell lineagesderived from cells of the EBs. Non-limiting examples of such culturingconditions are further described hereinunder.

Preferably, the method of this aspect of the present invention furtherincludes isolating lineage specific cells following step (b).

As used herein, the phrase “isolating lineage specific cells” refers tothe enrichment of a mixed population of cells in a culture with cellspredominantly displaying at least one characteristic associated with aspecific lineage phenotype. It will be appreciated that all celllineages are derived from the three embryonic germ layers. Thus, forexample, hepatocytes and pancreatic cells are derived from the embryonicendoderm, osseous, cartilaginous, elastic, fibrous connective tissues,myocytes, myocardial cells, bone marrow cells, vascular cells (namelyendothelial and smooth muscle cells), and hematopoietic cells aredifferentiated from embryonic mesoderm and neural, retina and epidermalcells are derived from the embryonic ectoderm.

According to one preferred embodiment of the present invention,isolating is effected by sorting of cells of the EBs via fluorescenceactivated cell sorter (FACS).

Methods of isolating EB-derived-differentiated cells via FACS analysisare known in the art. According to one method, EBs are disaggregatedusing a solution of Trypsin and EDTA (0.025% and 0.01%, respectively),washed with 5% fetal bovine serum (FBS) in phosphate buffered saline(PBS) and incubated for 30 min on ice with fluorescently-labeledantibodies directed against cell surface antigens characteristics to aspecific cell lineage. For example, endothelial cells are isolated byattaching an antibody directed against the platelet endothelial celladhesion molecule-1 (PECAM1) such as the fluorescently-labeled PECAM1antibodies (30884X) available from PharMingen (PharMingen, BectonDickinson Bio Sciences, San Jose, Calif., USA) as described inLevenberg, S. et al., (Endothelial cells derived from human embryonicstem cells. Proc. Natl. Acad. Sci. USA. 2002. 99: 4391-4396).Hematopoietic cells are isolated using fluorescently-labeled antibodiessuch as CD34-FITC, CD45-PE, CD31-PE, CD38-PE, CD90-FITC, CD117-PE,CD15-FITC, class I-FITC, all of which IgG1 are available fromPharMingen, CD133/1-PE (IgG1) (available from Miltenyi Biotec, Auburn,Calif.), and glycophorin A-PE (IgG1), available from Immunotech (Miami,Fla.). Live cells (i.e., without fixation) are analyzed on a FACScan(Becton Dickinson Bio Sciences) by using propidium iodide to excludedead cells with either the PC-LYSIS or the CELLQUEST software. It willbe appreciated that isolated cells can be further enriched usingmagnetically-labeled second antibodies and magnetic separation columns(MACS, Miltenyi) as described by Kaufman, D. S. et al., (Hematopoieticcolony-forming cells derived from human embryonic stem cells. Proc.Natl. Acad. Sci. USA. 2001, 98: 10716-10721).

According to yet an additional preferred embodiment of the presentinvention, isolating is effected by a mechanical separation of cells,tissues and/or tissue-like structures contained within the EBs.

For example, beating cardiomyocytes can be isolated from EBs asdisclosed in U.S. Pat. Appl. No. 20030022367 to Xu et al. Four-day-oldEBs of the present invention are transferred to gelatin-coated plates orchamber slides and are allowed to attach and differentiate.Spontaneously contracting cells, which are observed from day 8 ofdifferentiation, are mechanically separated and collected into a 15-mLtube containing low-calcium medium or PBS. Cells are dissociated usingCollagenase B digestion for 60-120 minutes at 37° C., depending on theCollagenase activity. Dissociated cells are then resuspended in adifferentiation KB medium (85 mM KCl, 30 mM K₂HPO₄, 5 mM MgSO₄, 1 mMEGTA, 5 mM creatine, 20 mM glucose, 2 mM Na₂ATP, 5 mM pyruvate, and 20mM taurine, buffered to pH 7.2, Maltsev et al., Circ. Res. 75:233, 1994)and incubated at 37° C. for 15-30 min. Following dissociation cells areseeded into chamber slides and cultured in the differentiation medium togenerate single cardiomyocytes capable of beating.

According to still an additional preferred embodiment of the presentinvention, isolating is effected by subjecting the EBs todifferentiation factors to thereby induce differentiation of the EBsinto lineage specific differentiated cells.

Following is a non-limiting description of a number of procedures andapproaches for inducing differentiation of EBs to lineage specificcells.

To differentiate the EBs of the present invention into neuralprecursors, four-day-old EBs are cultured for 5-12 days in tissueculture dishes including DMEM/F-12 medium with 5 mg/ml insulin, 50 mg/mltransferrin, 30 nM selenium chloride, and 5 mg/ml fibronectin (ITSFnmedium, Okabe, S. et al., 1996, Mech. Dev. 59: 89-102). The resultantneural precursors can be further transplanted to generate neural cellsin vivo (Brüstle, O. et al., 1997. In vitro-generated neural precursorsparticipate in mammalian brain development. Proc. Natl. Acad. Sci. USA.94: 14809-14814). It will be appreciated that prior to theirtransplantation, the neural precursors are trypsinized and triturated tosingle-cell suspensions in the presence of 0.1% DNase.

EBs of the present invention can differentiate to oligodendrocytes andmyelinate cells by culturing the cells in modified SATO medium, i.e.,DMEM with bovine serum albumin (BSA), pyruvate, progesterone,putrescine, thyroxine, triiodothryonine, insulin, transferrin, sodiumselenite, amino acids, neurotrophin 3, ciliary neurotrophic factor andHepes (Bottenstein, J. E. & Sato, G. H., 1979, Proc. Natl. Acad. Sci.USA 76, 514-517; Raff, M. C., Miller, R. H., & Noble, M., 1983, Nature303: 390-396]. Briefly, EBs are dissociated using 0.25% Trypsin/EDTA (5min at 37° C.) and triturated to single cell suspensions. Suspendedcells are plated in flasks containing SATO medium supplemented with 5%equine serum and 5% fetal calf serum (FCS). Following 4 days in culture,the flasks are gently shaken to suspend loosely adhering cells(primarily oligodendrocytes), while astrocytes are remained adhering tothe flasks and further producing conditioned medium. Primaryoligodendrocytes are transferred to new flasks containing SATO mediumfor additional two days. Following a total of 6 days in culture,oligospheres are either partially dissociated and resuspended in SATOmedium for cell transplantation, or completely dissociated and a platedin an oligosphere-conditioned medium which is derived from the previousshaking step [Liu, S. et al., (2000). Embryonic stem cells differentiateinto oligodendrocytes and myelinate in culture and after spinal cordtransplantation. Proc. Natl. Acad. Sci. USA. 97: 6126-6131].

For mast cell differentiation, two-week-old EBs of the present inventionare transferred to tissue culture dishes including DMEM mediumsupplemented with 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin,100 mg/ml streptomycin, 20% (v/v) WEHI-3 cell-conditioned medium and 50ng/ml recombinant rat stem cell factor (rrSCF, Tsai, M. et al., 2000. Invivo immunological function of mast cells derived from embryonic stemcells: An approach for the rapid analysis of even embryonic lethalmutations in adult mice in vivo. Proc Natl Acad Sci USA. 97: 9186-9190).Cultures are expanded weekly by transferring the cells to new flasks andreplacing half of the culture medium.

To generate hemato-lymphoid cells from the EBs of the present invention,2-3 days-old EBs are transferred to gas-permeable culture dishes in thepresence of 7.5% CO₂ and 5% O₂ using an incubator with adjustable oxygencontent. Following 15 days of differentiation, cells are harvested anddissociated by gentle digestion with Collagenase (0.1 unit/mg) andDispase (0.8 unit/mg), both are available from F. Hoffman-La Roche Ltd,Basel, Switzerland. CD45-positive cells are isolated using anti-CD45monoclonal antibody (mAb) M1/9.3.4.HL.2 and paramagnetic microbeads(Miltenyi) conjugated to goat anti-rat immunoglobulin as described inPotocnik, A. J. et al., (Immunology Hemato-lymphoid in vivoreconstitution potential of subpopulations derived from in vitrodifferentiated embryonic stem cells. Proc. Natl. Acad. Sci. USA. 1997,94: 10295-10300). The isolated CD45-positive cells can be furtherenriched using a single passage over a MACS column (Miltenyi).

It will be appreciated that the culturing conditions suitable for thedifferentiation and expansion of the isolated lineage specific cellsinclude various tissue culture media, growth factors, antibiotic, aminoacids and the like and it is within the capability of one skilled in theart to determine which conditions should be applied in order to expandand differentiate particular cell types and/or cell lineages.

Additionally or alternatively, lineage specific cells can be obtained bydirectly inducing the expanded, undifferentiated ESCs to culturingconditions suitable for the differentiation of specific cell lineage.

In addition to the lineage-specific primary cultures, EBs of the presentinvention can be used to generate lineage-specific cell lines which arecapable of unlimited expansion in culture.

Cell lines of the present invention can be produced by immortalizing theEB-derived cells by methods known in the art, including, for example,expressing a telomerase gene in the cells (Wei, W. et al., 2003. MolCell Biol. 23: 2859-2870) or co-culturing the cells with NIH 3T3hph-HOX11 retroviral producer cells (Hawley, R. G. et al., 1994.Oncogene 9: 1-12).

It will be appreciated that since the lineage-specific cells or celllines obtained according to the teachings of the present invention aredeveloped by differentiation processes similar to those naturallyoccurring in the human embryo they can be further used for humancell-based therapy and tissue regeneration.

Thus, the present invention envisages the use of the expanded and/ordifferentiated lineage-specific cells or cell lines of the presentinvention for treating a disorder requiring cell replacement therapy.

For example, oligodendrocyte precursors can be used to treat myelindisorders (Repair of myelin disease: Strategies and progress in animalmodels. Molecular Medicine Today. 1997. pp. 554-561), chondrocytes ormesenchymal cells can be used in treatment of bone and cartilage defects(U.S. Pat. No. 4,642,120) and cells of the epithelial lineage can beused in skin regeneration of a wound or burn (U.S. Pat. No. 5,716,411).

For certain disorders, such as genetic disorders in which a specificgene product is missing [e.g., lack of the CFTR gene-product in cysticfibrosis patients (Davies J C, 2002. New therapeutic approaches forcystic fibrosis lung disease. J. R. Soc. Med. 95 Suppl 41:58-67)],ESC-derived cells are preferably manipulated to over-express the mutatedgene prior to their administration to the individual. It will beappreciated that for other disorders, the ESC-derived cells should bemanipulated to exclude certain genes.

Over-expression or exclusion of genes can be effected using knock-inand/or knock-out constructs [see for example, Fukushige, S. and Ikeda,J. E.: Trapping of mammalian promoters by Cre-lox site-specificrecombination. DNA Res 3 (1996) 73-50; Bedell, M. A., Jerkins, N. A. andCopeland, N. G.: Mouse models of human disease. Part I: Techniques andresources for genetic analysis in mice. Genes and Development 11 (1997)1-11; Bermingham, J. J., Scherer, S. S., O'Connell, S., Arroyo, E.,Kalla, K. A., Powell, F. L. and Rosenfeld, M. G.: Tst-1/Oct-6/SCIPregulates a unique step in peripheral myelination and is required fornormal respiration. Genes Dev 10 (1996) 1751-62].

In addition to cell replacement therapy, the lineage specific cells ofthe present invention can also be utilized to prepare a cDNA library.mRNA is prepared by standard techniques from the lineage specific cellsand is further reverse transcribed to form cDNA. The cDNA preparationcan be subtracted with nucleotides from embryonic fibroblasts and othercells of undesired specificity, to produce a subtracted cDNA library bytechniques known in the art.

The lineage specific cells of the present invention can be used toscreen for factors (such as small molecule drugs, peptides,polynucleotides, and the like) or conditions (such as culture conditionsor manipulation) that affect the differentiation of lineage precursor toterminally differentiated cells. For example, growth affectingsubstances, toxins or potential differentiation factors can be tested bytheir addition to the culture medium.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., Ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., Ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., Ed. (1994); Stites et al.(Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., Ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,Eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., Ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Culturing Human Embryonic Stem Cell with a Medium Containingthe IL-6-IL-6 Receptor (IL6RIL6) Chimera

To optimize culturing conditions of hESCs on feeder-layer free culturingsystems, the present inventors have tested various combinations ofgrowth factors, as follows.

Materials and Experimental Methods

hESC Culture—

Human embryonic stem cell (hESC) lines I-6 and I-3 [Amit &Itskovitz-Eldor, 2002] were cultured for 46 and 39 passagesrespectively, with inactivated mouse embryonic fibroblasts (MEFs) in an“hESC basic culture medium” consisting of 85% Ko-DMEM, supplemented with15% serum replacement (SR), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol,1% non-essential amino acid stock, and 4 ng/ml bFGF (all GibcoInvitrogen Corporation products, Grand Island N.Y., USA).

To test various media combinations on feeder-layer free culturingsystems, hESCs were transferred to 50 μg per 10 cm² fibronectin-coveredplates (human plasma fibronectin, Chemicon International, TemeculaCalif., USA) in the presence of the “hESC basic culture medium” with thefollowing combinations of cytokines:

(i) “CM6 medium”—0.3 ng/ml Interleukin-6 (IL6) and 0.5 ng/ml IL6 solublereceptor (both from R&D Systems Minneapolis Minn., USA);

(ii) “IL-6-IL-6 receptor (IL6RIL6) chimera”—50 ng/ml, 100 ng/ml, 200ng/ml or 300 ng/ml of IL6RIL6 chimera (Chebath J, et al., 1997 and WO99/02552 to Revel M., et al. SEQ ID NO:31). When used with 100 ng/ml ofthe IL6RIL6 chimera, this medium is also called CM100.

(iii) Control medium—4 ng/ml bFGF (Gibco Invitrogen Corporation, GrandIsland N.Y., USA).

Cells were passaged every four to six days using 1 mg/ml type IVcollagenase (Gibco Invitrogen Corporation, Grand Island N.Y., USA).Cells were frozen in liquid nitrogen using a freezing solutionconsisting of 10% DMSO (Sigma, St Louis Mo., USA), 15% SR (ChemiconInternational, Temecula Calif., USA) and 75% Ko-DMEM (Gibco-InvitrogenCorporation, Grand Island N.Y., USA) [Amit et al, 2004].

Immunohistochemistry—

Undifferentiated hESCs grown in the feeder-free culture system anddifferentiated cells dissociated using trypsin-EDTA from 14-day-old EBswere fixed with 4% paraformaldehyde and incubated overnight at 4° C.with 1:50 dilutions of the following primary antibodies: stage-specificembryonic antigen (SSEA) 1 (SSEA-1), 3 (SSEA-3) and 4 (SSEA-4)(Hybridoma Bank, Iowa, USA), tumor recognition antigen (TRA) 1-60 andTRA1-81 (Chemicon International, Temecula Calif., USA) and Oct4 (SantaCruse). Cys 3 conjugated antibodies (Chemicon International, TemeculaCalif., USA) were used as secondary antibodies at a 1:100 dilution.

Karyotype Analysis—

Karyotype analysis (G-banding) was performed on at least 20 cells fromeach sample, two samples per test, as previously described [Amit et al,2003]. Karyotypes were analyzed and reported according to the“International System for Human Cytogenetic Nomenclature” (ISCN).

EB Formation—

For the formation of EBs, one to four confluent wells were used in asix-well plate (40 cm²). ESCs were removed from their culture dish using1 mg/ml type IV collagenase, further broken into small clumps using 1000μl Gilson pipette tips, and cultured in suspension in 58 mm petri dishes(Greiner, Frickenhausen, Germany). EBs were grown in medium consistingof 80% Ko-DMEM, supplemented with 20% FBSd (HyClone, Utah, USA), 1 mML-glutamine, 0.1 mM β-mercaptoethanol, and 1% non-essential amino acidstock (all but FBSd from Gibco Invitrogen Corporation, Grand IslandN.Y., USA). 10 day-old EBs were harvested for RNA isolation andhistological examination.

RT PCR Analysis—

Total RNA was isolated from hESCs grown for 10-15 passages infeeder-free conditions, or from 10 day-old EBs (formed from cells grownin the feeder-free system) using Tri-Reagent (Sigma, St. Louis Mo.,USA), according to the manufacturer's instructions. cDNA was synthesizedfrom 1 μg total RNA using MMLV reverse transcriptase RNase H minus(Promega, Madison Wis., USA). PCR reactions included denaturation for 5minutes at 94° C. followed by repeated cycles of 94° C. for 30 seconds,annealing for 30 seconds at a temperature specified in Table 1,hereinbelow, and extension at 72° C. for 30 seconds. PCR primers andreaction conditions used are described in Table 1, hereinbelow. PCRproducts were size-fractionated using 2% agarose gel electrophoresis.DNA markers were used to confirm the size of the resultant fragments.

TABLE 1  RT-PCR conditions Gene product (AccessionForward (F) and reverse (R) primers  Reaction Size number) (SEQ ID NO:)Condition (bp) Oct-4 F: 5′-GAGAACAATGAGAACCTTCAGGA (SEQ ID NO: 1)30 cycles 219 (S81255) R: 5′-TTCTGGCGCCGGTTACAGAACCA (SEQ ID NO: 2)at 60° C. in 1.5 mM MgCl₂ AlbuminF: 5′-TGCTTGAATGTGCTGATGACAGGG (SEQ ID NO: 3) 35 cycles 302 (AF542069)R: 5′-AAGGCAAGTCAGCAGCCATCTCAT (SEQ ID NO: 4) at 60° C. in 1.5 mM MgCl₂α-fetoprotein F: 5′-GCTGGATTGTCTGCAGGATGGGGAA (SEQ ID NO: 5) 30 cycles216 (BC027881) R: 5′-TCCCCTGAAGAAAATTGGTTAAAAT (SEQ ID NO: 6) at 60° C.in 1.5 mM MgCl₂ NF-68KD F: 5′-GAGTGAAATGGCACGATACCTA (SEQ ID NO: 7)30 cycles 473 (NFH R: 5′-TTTCCTCTCCTTCTTCACCTTC (SEQ ID NO: 8) at 60° C.(AY156690; in 2 mM X15307; MgCl₂ X15309) α-cardiacF: 5′-GGAGTTATGGTGGGTATGGGTC (SEQ ID NO: 9) 35 cycles 486 actinR: 5′-AGTGGTGACAAAGGAGTAGCCA (SEQ ID NO: 10) at 65° C. (NM_005159)in 2 mM MgCl₂ β-Actin F: 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG 35 cycles838 (NM_001101) (SEQ ID NO: 11) at 62° C.R: 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC in 1.5 mM (SEQ ID NO: 12) MgCl₂Sox2 5′ CCCCCGGCGGCAATAGCA (SEQ ID NO: 13) 35 cycles 448 (Z31560) 3′TCGGCGCCGGGGAGATACAT (SEQ ID NO: 14) at 60° C. in 1.5 mM MgCl₂ Rex1 5′GCGTACGCAAATTAAAGTCCAGA (SEQ ID NO: 15) 35 cycles 306 (AF450454) 3′CAGCATCCTAAACAGCTCGCAGAAT (SEQ ID NO: 16) at 56° C. in 1.5 mM MgCl₂ CX435′ TACCATGCGACCAGTGGTGCGCT (SEQ ID NO: 17) 35 cycles 295 (NM_000165) 3′GAATTCTGGTTATCATCGGGGAA (SEQ ID NO: 18) at 61° C. in 1.5 mM MgCl₂ FGF45′ CTACAACGCCTACGAGTCCTACA (SEQ ID NO: 19) 35 cycles 370 (NM_002007) 3′GTTGCACCAGAAAAGTCAGAGTTG (SEQ ID NO: 20) at 52° C. in 1.5 mM MgCl₂Glucagon 5′ CTCAGTGATCCTGATCAGATGAACG (SEQ ID NO: 21) 35 cycles 370(X03991) 3′ AGTCCCTGGCGGCAAGATTATCAAG (SEQ ID NO: 22) at 65° C.in 1.5 mM MgCl₂ β-globulin 5′ ACCTGACTCCTGAGGAGAAGTCTGC (SEQ ID NO: 23)35 cycles 410 (V00499) 3′ TAGCCACACCAGCCACCACTTTCTG (SEQ ID NO: 24)at 65° C. in 1.5 mM MgCl₂ Flk1 5′ ATGCACGGCATCTGGGAATC (SEQ ID NO: 25)35 cycles 537 (NM_002253) 3′ GCTACTGTCCTGCAAGTTGCTGTC (SEQ ID NO: 26)at 65° C. in 1.5 mM MgCl₂ AC133 5′ CAGTCTGACCAGCGTGAAAA (SEQ ID NO: 27)35 cycles 200 (NM_006017) 3′ GGCCATCCAAATCTGTCCTA (SEQ ID NO: 28) at 65°C. in 1.5 mM MgCl₂ Nanog 5′ ACTAACATGAGTGTGGATCC (SEQ ID NO: 29)35 cycles 800 (NG_004095) 3′ TCATCTTCACACGTCTTCAG (SEQ ID NO: 30) at 61°C. in 1.5 mM MgCl₂ Table 1: RT-PCR primers and PCR conditions areprovided along with the GenBank Accession numbers of the amplifiedtranscripts.

Experimental Results

Medium Supplemented with a Combination of 100 ng/ml IL6RIL6 Chimera and4 ng/ml bFGF is Most Suitable to Support Undifferentiated Proliferationof hESCs on a Feeder-Free Culture System—

Several concentrations of the IL6RIL6 chimera were tested for theability to support the feeder-layer free culture of hESCs. Initially,two measures were used to estimate the ability of the hESCs to grow inthe feeder-free culture system, namely percentage of differentiation andrate of growth. The medium supplemented with the combination of 100ng/ml IL6RIL6 chimera and 4 ng/ml bFGF was found to be the most suitableto support undifferentiated feeder-layer free hESC proliferation.Culturing hESCs with a culture medium consisting of 4 ng/ml bFGF and thechimera components IL-6 and IL-6 soluble receptor failed to support hESCprolonged culture and resulted in differentiation of hESCs within 3-5passages (data not shown). Although 70% of the hESCs remained in theundifferentiated stage in these conditions for a few passages, theproliferation rate was low and with each passage the number of survivinghESCs decreased. In addition, culturing of hESCs in a medium containingthe IL6RIL6 chimera and serum replacement, in the absence of bFGF,resulted in low proliferation of cells and failure to maintain the hESCsbeyond 1-2 passages (data not shown). However, in medium supplementedwith either 100 ng/ml or 300 ng/ml IL6RIL6 chimera and 4 ng/ml bFGF, thehESCs could be cultured continuously in feeder-layer free conditions forat least 43 passages. hESCs cultured in these conditions (in thepresence of the IL6RIL6 chimera medium) maintained their ESC features,including undifferentiated proliferation, karyotype stability andpluripotency as was tested following 28 passages. Based on morphology,the background spontaneous differentiation rates were about 15%, similarto those occurring in other feeder layer-free culture methods [Xu et al,2001, 2005; Amit et al, 2004]. Examples of undifferentiated coloniescultured in feeder-layer free conditions are illustrated in FIGS. 1a-1f. Interestingly, when cultured in a medium supplemented with the IL6RIL6chimera and bFGF using fibronectin as substrate for the feeder-freeculture system, the cells differentiated at the periphery of thecolonies and formed an outgrowth of feeder-like cells (FIG. 1a ). Insimilar cultures, laminin did not induce such differentiation (FIG. 1c )but, human and mouse laminin matrices were found to be inconsistent intheir ability to support hESC undifferentiated growth and, therefore,fibronectin appears preferable despite the formation of feeder-likecells. Former studies on feeder-layer free cultures of hESCs alsoreported the formation of feeder-like cells [Xu et al, 2001].

Feeder-Free Culture System in the Presence of a Culture MediumContaining the IL6RIL6 Chimera Supports hESCs with Typical Morphology—

When compared to hESCs cultures on exogenous feeder layers, nomorphological differences could be observed between colonies grown inthe feeder layer-free culture system (a fibronectin matrix and a mediumsupplemented with IL6RIL6 chimera and bFGF) and those grown on MEFs(FIGS. 1a-1f and data not shown). Correspondingly, the morphologicalfeatures at the single-cell level remained unchanged, the cells remainedsmall and round, and exhibited high nucleus-to-cytoplasm ratio, with anotable presence of one to three nucleoli and with typical spacingbetween the cells (FIGS. 1a-1f ). The hESC population doubling time wassimilar to that observed when grown on MEFs (about 36 hours), and in thepresent feeder-free conditions the cells could be passaged routinelyevery four to six days, at a ratio of 1/2 or 1/3, similar to thesplitting ratio employed when the hESCs are cultured on MEFs. The cellswere passaged at the same seeding efficiency as with MEF of about 1million cells per 10 cm², with the same viability rate of over 90%.Using 15% SR and 10% DMSO, cells were successfully frozen and thawed.

The Fibronectin Feeder-Layer Free Culture System Supplemented with aMedium Containing the IL6RIL6 Chimera Supports hESCs with NormalKaryotype for at Least 23 Passages—

Karyotype analysis by Giemsa banding was carried out on two separatecultures, representing the two medium conditions: medium supplementedwith 4 ng/ml bFGF and either 100 ng/ml or 300 ng/ml IL6RIL6 chimera 23passages after transferring the cells into the feeder-layer environmentwhich is based on fibronectin. At least 20 cells were tested from eachsample, 40 cells from each medium combination. All examined cells werefound to sustain normal karyotype of 46, XX and 46,XY for cell lines I3and I6, respectively. Examples of chromosomes from examined cells areillustrated in FIG. 2. Overall, these results demonstrate that thecells' karyotype remains stable in the fibronectin based feeder freeculture system supplemented with the IL6RIL6 chimera medium, similarlyto ESCs grown with MEFs.

hESCs Cultured on Fibronectin Feeder Free Culture Systems Supplementedwith the IL6RIL6 Chimera Medium Express Typical hESC Markers ofUndifferentiated Cells—

Several surface markers typical of primate undifferentiated ES cellswere examined using immunofluorescent staining as described elsewhere[Thomson et al, 1995, 1996, 1998]. hESCs cultured on the fibronectinfeeder-free culture system for 27 passages with medium supplemented with100 ng/ml or 300 ng/ml of the IL6RIL6 chimera were found to be stronglypositive to surface markers TRA-1-60 (FIG. 3a ) Oct4 (FIG. 3b ), SSEA4(FIG. 3c ), and TRA-1-81 (data not shown). As in other primate ES cells,staining with SSEA3 was weak and negative for SSEA1 (data not shown).

The IL6RIL6 Chimera Medium is Capable of Supporting Pluripotent hESCs asEvident by the In Vitro Differentiation into Embryoid Bodies (EBs)—

The developmental potential of the cells after prolonged culture infeeder layer-free conditions was examined in vitro by the formation ofEBs. When cultured in suspension, after 7 passages in mediumsupplemented with either 100 ng/ml or 300 ng/ml IL6RIL6 chimera, hESCsformed EBs similar to those created by ES cells grown on MEFs. Withinthese EBs, stem cells differentiated into cell types representative ofthe three embryonic germ layers.

EBs Formed from the hESCs Cultured on the IL6RIL6 Chimera ExpressedTypical Differentiation Markers of all Embryonic Cell Lineages—

To further define the expression pattern of the undifferentiated hESCsgrown on the fibronectin-based feeder free culture system in thepresence of a medium containing the IL6RIL6 chimera, RT-PCR analysis wasperformed using primers specific to both differentiation andundifferentiated cells (primers employed are listed in Table 1,hereinabove). As is shown in FIGS. 4a-4l , while undifferentiated cellscultured on the fibronectin feeder free culture system using mediumsupplemented with the IL6RIL6 chimera expressed undifferentiated geneticmarkers such as Oct 4 (FIG. 4a ), Nanog (data not shown), Sox2 (FIG. 4b), Rex1 (FIG. 4c ), Cx43 (FIG. 4d ) and FGF4 (FIG. 4e ) [Bhattacharya etal, 2004], cells harvested from 10 day-old EBs expressed genes such asalbumin (FIG. 4f ) and glucagon (FIG. 4g ) (typical of the embryonicendoderm), α-cardiac actin (FIG. 4i ), β-globulin (FIG. 4h ) and Flk1(FIG. 4j ) (typical of the embryonic mesoderm), and AC133 (FIG. 4k ) andneurofilament (NFH; FIG. 4l ) (typical of the embryonic ectoderm).

Altogether, these results demonstrate the identification of a definedmedium based on the IL6RIL6 chimera which is suitable for maintaininghESCs in a pluripotent and undifferentiated state on a feeder-freeculture system such as the fibronectin based feeder free system.

Analysis and Discussion

Human ESCs, like mouse ES cells, are traditionally cultured with MEFs,which may expose them to animal pathogens. In this study, the presentinventors have demonstrated a defined medium and feeder layer-freeculture system based on the use of serum Replacement™, bFGF, IL6RIL6chimera and human fibronectin as a matrix substitute.

Several concentrations of the IL6RIL6 chimera were tested for theirability to maintain hESCs in an undifferentiated state. The mostsuitable medium was that supplemented with 100 ng/ml of the IL6RIL6chimera together with 4 ng/ml bFGF, in which the three transferred hESClines continued to proliferate while retaining normal hESC featuresthroughout the prolonged culture.

Cells cultured in these conditions maintained all characteristics of EScells. After prolonged culture of up to 28 passages, the cells remainedundifferentiated, as demonstrated by the colony and single cellmorphology, and by the expression of surface markers typical ofundifferentiated primate ESCs [Thomson et al, 1995, 1996, 1998;Reubinoff et al, 2000]. In addition, while cultured in these conditions,hESCs expressed specific markers for the undifferentiated stage such asOct 4, Sox 2, Rex1 and FGF4, as demonstrated by immunofluorescence (forOct4) and RT-PCR analyses (for Oct4, Sox 2, Rex1 and FGF4).

Karyotype analysis carried out on representative cell samplesdemonstrated that the hESCs' karyotype remained stable in the proposedconditions (after 23 passages at the tested conditions). None of theexamined 40 cells exhibited karyotype abnormalities.

The cells' pluripotency was examined in vitro. Cells cultured in thefeeder layer-free culture system for more than 10 passages, formed EBssimilar to those created when grown on MEFs [Itskovitz-Eldor et al,2000]. RT-PCR analysis demonstrated that cells within these EBsdifferentiated into different cell types representative of the threeembryonic germ layers.

These results demonstrate that hESCs can be maintained asundifferentiated cells in defined feeder- and serum-free conditionswhich include the IL6RIL6 chimera while exhibiting hESC features.

The mechanism by which hESCs self-maintain is still not clear. In mouseESCs the role of LIF and other members of the IL-6 family, actingthrough gp130 and the JAK/STAT3 pathway, in maintaining ESCsundifferentiated prolonged culture is well known [Smith et al, 1988;Williams et al, 1988; Rose et al, 1994; Conover et al, 1993; Niwa et al,1998]. Furthermore, to date the only method for deriving new mouse ESClines in feeder layer-free conditions is based on the addition offactors from the IL-6 family [Nichols et al, 1994]. The IL6RIL6 chimerawas demonstrated as the most potent factor in supporting the feederlayer-free isolation of mouse ESC lines (Nichols et al, 1994). Previousstudies did not demonstrate a significant effect of the IL-6 family,including a fusion protein of portions of IL-6 and the IL-6 receptor, onthe self-maintenance of undifferentiated hESCs [Daheron et al, 2004;Humphrey et al, 2004; Sato et al, 2004]. The specific conditions forhESC cultures described here may explain why the IL6RIL6 chimera waseffective in supporting the proliferation of hESC with minimaldifferentiation. One difference being the use of fibronectin substrateas a matrix (and not mouse laminin) and another being the preciseschedule of hESC passaging. Further research is required to elucidatethe underlying mechanisms of action of the IL6RIL6 chimera at the levelof signal transduction, its time course and intensity at which differentpathways (JAK/STAT, PI3 kinase, MAPK, see Hirano et al, 1997) areactivated.

Future clinical uses of hESCs will require a reproducible, well-definedand xeno-free culture system. Although the serum replacement (SR) usedin the present study is considered such, it contains “Albumax” which isa lipid-enriched bovine serum albumin and, therefore, is notanimal-free. The well-defined conditioned media demonstrated in thepresent study are suitable for culturing hESCs and may be advantageousfor undertaking research on the mechanisms of ESC self-maintenance,especially of the possible roles of the LIF/STAT3 pathway and variousintegrins as fibronectin receptors. Other studies using hESCs, such asthe research on differentiation pathways and mechanisms, will benefitfrom the availability of a well-defined and reproducible culture system.

This culture system is a further step forward towards fully definedculture conditions for hESCs, and promotes the development of axeno-free culture system for hESCs.

Example 2 Culturing Human Embryonic Stem Cell with a Medium ContainingTGF-Beta Isoforms Devoid of Serum, Serum Replacement and Albumin

Materials and Experimental Methods

ESC Culture—

Human embryonic stem cell (hESC) lines I-6, I4 and I-3 [Amit &Itskovitz-Eldor, 2002] were cultured with inactivated mouse embryonicfibroblasts (MEFs) for 40-60 passages in a “basic hESC culture medium”consisting of 85% DMEM/F12 (Biological Industries, Biet Haemek, Israel)supplemented with 15% serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock, and 4 ng/ml basicfibroblast growth factor (bFGF) (all Gibco Invitrogen Corporationproducts, Grand Island N.Y., USA).

To test the ability of various culture media to support the growth ofhESC in an undifferentiated yet pluripotent state the hESCs weretransferred to several culture systems:

(i) Fibronectin feeder-free culture system—50 μg per 10 cm²fibronectin-covered plates (human plasma fibronectin, ChemiconInternational, Temecula Calif., USA);

(ii) Matrigel™ feeder-free culture system—Matrigel™ (BD Biosciences,Bedford, Mass., USA);

(iii) MEFs—mouse embryonic fibroblast feeder layer system;

(iv) Foreskins fibroblasts—foreskin fibroblasts feeder layer system.

Tested Media—

The tested medium were based on:

(i) D1 medium—Mab ADCB medium (HyClone, Utah, USA) supplemented with 2mM L-glutamine (Invitrogen Corporation products, Grand Island N.Y.,USA), 0.12 ng/ml TGFβ₁ (from R&D Systems Minneapolis Minn., USA), and 10ng/ml bFGF (Invitrogen Corporation products, Grand Island N.Y., USA).

(ii) D2 medium—Mab ADCB medium (HyClone, Utah, USA) supplemented with 2mM L-glutamine (Invitrogen Corporation products, Grand Island N.Y.,USA), 2 ng/ml TGFβ₃ and 10 ng/ml bFGF (Invitrogen Corporation products,Grand Island N.Y., USA).

(iii) HA16 medium—96% DMEM/F12 (Biological Industries, Biet Haemek,

Israel) supplemented with 1:1000 dilution of the ITS Premix [the ITSpremix is a ×1000 stock solution obtained from BD Biosciences, Bedford,Mass., USA and consists of 12.5 mg Insulin, 12.5 mg Transferrin and 12.5mg Selenius acid], 2 mM L-glutamine, 2 ng/ml TGFβ₃ (from R&D SystemsMinneapolis Minn., USA), 4 ng/ml bFGF, 500 ng/ml ascorbic acid (Sigma,Steinheim, Germany), and a 1:1000 dilution of a lipid mixture (SigmaCat. No. L5146, Steinheim, Germany) (all but those otherwise specifiedwere obtained from Gibco Invitrogen Corporation products, Grand IslandN.Y., USA).

(iv) HA19 medium—96% DMEM/F12 (Biological Industries, Beth Haemek,Israel) supplemented with 1:1000 dilution of the ITS premix (BDBiosciences, Bedford, Mass., USA), 2 mM L-glutamine, 2 ng/ml TGFβ₃ (fromR&D Systems Minneapolis Minn., USA), 4 ng/ml bFGF, 500 ng/ml ascorbicacid (Sigma, Steinheim, Germany), a 1:1000 dilution of a lipid mixture(Sigma Cat. No. L5146, Steinheim, Germany) and a 1:100 dilution ofSimfronic 68 (Pluronic F-68 solution, P5556 from Sigma, Steinheim,Germany, the stock is 10%, the F-68 in culture is provided at aconcentration of 0.1%) (Sigma, Steinheim, Germany) (all but thoseotherwise specified were obtained from Gibco Invitrogen Corporationproducts, Grand Island N.Y., USA).

(v) CM100F medium—85% DMEM/F12 (Biological Industries, Biet Haemek,Israel) supplemented with 15% serum replacement (SR), 2 mM L-glutamine,0.1 mM β-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/mlbasic fibroblast growth factor (bFGF) and 100 ng/ml IL6RIL6 chimera (SEQID NO:31, a kind gift from Prof. Revel M, the Weizmann Inst. Rehovot,Israel) (all but those otherwise specified were obtained from GibcoInvitrogen Corporation products, Grand Island N.Y., USA). As a control,the same culture media was used with the removal of the growth factors(except for bFGF which remained in the control culture medium) and theIL6RIL6 chimera.

(vi) “IL6-IL-6 receptor (IL6RIL6) chimera”—85% Ko-DMEM, supplementedwith 15% serum replacement (SR), 2 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock, 4 ng/ml bFGF and50 ng/ml, 100 ng/ml, 200 ng/ml or 300 ng/ml of IL6RIL6 chimera (ChebathJ, et al., 1997 and WO 99/02552 to Revel M., et al. SEQ ID NO:31) (allGibco Invitrogen Corporation products, Grand Island N.Y., USA). Whenused with 100 ng/ml of the IL6RIL6 chimera, this medium is also calledCM100.

(vii) HACM100 medium—96% DMEM/F12 (Biological Industries, Biet Haemek,Israel) supplemented with a 1:1000 dilution of the ITS premix (BDBiosciences, Bedford, Mass., USA), 2 mM L-glutamine, 4 ng/ml bFGF, 500ng/ml ascorbic acid (Sigma, Steinheim, Germany), a 1:1000 dilution of alipid mixture (Sigma Cat. No. L5146, Steinheim, Germany) and 100 ng/mlof IL6RIL6 chimera.

Cells were passaged every four to six days using 1.5 mg/ml type IVcollagenase (Worthington biochemical corporation, Lakewood, N.J., USA).Cells were frozen in liquid nitrogen using a freezing solutionconsisting of 10% DMSO (Sigma, St Louis Mo., USA), 40% human serum(HyClone, Utah, USA) and 50% DMEM/F12 (Biological Industries, BeitHaemek, Israel).

Derivation of New hESC Lines

Blastocyst Cultivation—

Zygotes were donated by couples undergoing pre-implantation geneticdiagnosis (PGD) or in vitro fertilization (IVF) at Cornell MedicalCollege, N.Y., who signed informed consent forms. The couples underwentthe traditional IVF procedure after ovarian stimulation withgonadotropins and oocyte retrieval. Zygotes were cultured to theblastocyst stage according to IVF laboratory standard protocol: underoil using specialized C1/C2 media for insemination, growth andblastocyst development (Cornell).

Derivation of hESC Lines—

After digestion of the zona pellucida by Tyrode's acidic solution(Sigma, St Louis Mo., USA) or its mechanical removal, the exposedblastocysts were placed in whole on a mitotically inactivated foreskinfibroblasts feeder layer (line F21 which was cultured in an animal freemedium since its derivation until used). For the derivation and initialpassages, cells were grown in the D2 or HA16 culture medium. The cellswere initially passaged mechanically every four to ten days.

Immunohistochemistry—

Undifferentiated hESCs grown in the tested culture system were fixedwith 4% paraformaldehyde and exposed to the primary antibodies (1:50)overnight at 4° C. Stage-specific embryonic antigen (SSEA) 1, 3 and 4(Hybridoma Bank, Iowa, USA), tumor recognition antigen (TRA) 1-60 andTRA1-81 (Chemicon International, Temecula Calif., USA) and Oct 4 (SantaCruz Biotechnology, Santa Cruz, Calif., USA) were used as primaryantibodies. Cys 3 conjugated antibodies (Chemicon International,Temecula Calif., USA) were used as secondary antibodies (1:200);

Karyotype Analysis—

Karyotype analysis (G-banding) was performed on at least 20 cells fromeach sample, two samples per test, as previously described [Amit et al,2003]. Karyotypes were analyzed and reported according to the“International System for Human Cytogenetic Nomenclature” (ISCN).

EB Formation—

For the formation of EBs, one to three confluent wells were used in asix-well plate (30 cm²). ESCs were removed from their culture dish using1 mg/ml type IV collagenase, further broken into small clumps using 1000μl Gilson pipette tips, and cultured in suspension in 58 mm petri dishes(Greiner, Frickenhausen, Germany). EBs were grown in differentiationmedium consisting of 80% DMEM/F12 (Biological Industries, Beit Haemek,Israel), supplemented with 20% FBSd (HyClone, Utah, USA), and 1 mML-glutamine (Invitrogen Corporation, Grand Island N.Y., USA).

RT PCR—

Total RNA was isolated from hESCs grown for over 10 passages infeeder-free conditions, or from 10 day-old EBs (created from cells grownin the tested culture system for more then 10 passages) usingTri-Reagent (Sigma, St. Louis Mo., USA), according to the manufacturer'sinstructions. cDNA was synthesized from 1 μg total RNA using MMLVreverse transcriptase RNase H minus (Promega, Madison Wis., USA). PCRreaction included denaturation for 5 minutes at 94° C. followed byrepeated cycles of 94° C. for 30 seconds, annealing for 30 seconds at atemperature as specified in Table 1 and extension at 72° C. for 30seconds. PCR primers and reaction conditions used are described in Table1 (see Example 1, hereinabove). PCR products were size-fractionatedusing 2% agarose gel electrophoresis. DNA markers were used to confirmthe size of the resultant fragments.

Teratoma Formation—

For teratoma formation, cells cultured in the offered culture methodsfor more than 15 passages, were injected into the rear leg muscle of4-week-old male SCID-beige mice (two mice for each tested culturesystem). Cell numbers ranged from 5×10⁶ cells to 10⁷ cells perinjection. Three to eight to 12 weeks after injection the mice weresacrificed and the resulting teratomas examined histologically.

Experimental Results

In this study the ability of few medium combinations, HA16, HA19, D1,D2, and CM100 to support undifferentiated and prolonged culture of hESCsin different culture conditions was examined. The basic medium, D1 orD2, is a commercial medium design for industrial and clinical proposesfor the culture of hybridomas in suspension. The medium is free fromanimal, serum products and proteins. HA16 and HA19 are based on definedmaterials only.

The effect of two isoforms of TGFβ, TGFβ₁ and TGFβ₃, in supporting hESCsundifferentiated culture, was examined. Initially, two measures wereused to estimate the ability of hESCs to grow in several culturesystems, namely percentage of differentiation and rate of growth. Theculture system used were: (1) feeder layer-free culture system based onfibronectin or Matrigel™ which are the most used matrices; (2) MEF, and(3) foreskin fibroblast. Based on these two parameters, the mediasupplemented with TGFβ₃, D2, HA16 and HA19, were found to be the mostsuitable to support undifferentiated hESC proliferation in all testedculture methods. Culture medium supplemented with 10 ng/ml bFGF only,failed to support hESC prolonged culture, in all the tested cultureconditions. Although 60% of the hESCs remained at the undifferentiatedstage in these conditions for a few passages, the proliferation rate waslow and with each passage the number of surviving hESCs decreased andthe percentage of background differentiation was increased.

D1 Medium on a Feeder Layer-Free System is Capable of Maintaining allhESCs Features Along with High Proliferation Rate—

When cultured in the feeder layer-free systems in the presence of the D1medium, which is supplemented with TGFβ₁, the hESCs maintained all hESCsfeatures including high proliferation rates. When cultured on the testedfeeder layers in the presence of the D1 medium, the hESCs demonstrated arelatively high background differentiation rate of 20% and lowproliferation abilities as compared to hESCs cultured at the same feederlayers systems with the D2 HA19 or HA16 medium.

D1, D2 and HA16 Media in Feeder Layer-Free are Capable of MaintaininghESCs in a Proliferative, Undifferentiated State, with ChromosomalStability and Pluripotency—

Human ESCs grown in the presence of the D1, D2 or HA16 medium infeeder-layer free conditions were cultured continuously for up to 53, 24or 10 passages, respectively, while maintaining their ESC features,including undifferentiated proliferation, chromosomal stability (astested by karyotype analysis, not shown) and pluripotency. Thebackground differentiation rates were found to be less than 10%, whichis similar to the differentiation rates occurring when hESCs arecultured in the traditional culture system based on MEFs as the feederlayer and medium supplemented with serum replacement and 4 ng/ml bFGF[Amit et al, 2000]. Examples of undifferentiated colonies cultured withD1, D2 or HA16 medium in feeder-layer free conditions and with the D2 orHA16 medium with the tested feeder layers are illustrated in FIGS. 5a -5d.

hESCs Cultured on Feeder Layer-Free Systems in the Presence of the D1 orthe D2 Medium are Devoid of Autofeeder—

Interestingly, when the hESCs were cultured in either the D1 or D2medium on the feeder layer-free system the cells did not differentiateat the periphery of the colonies and did not form an outgrowth offeeder-like cells (also called “autofeeder”) (FIG. 5d ), as described inother reports on feeder layer-free culture methods for hESCs (Xu et al,2001). No morphological differences could be observed between coloniesgrown in the feeder layer-free culture system and those grown withfeeder layers (FIGS. 5a-5d ). Correspondingly, morphological featuresremained unchanged on a single-cell level, rendering cells small andround, and exhibiting high nucleus-to-cytoplasm ratio, with a notablepresence of one to three nucleoli and typical spacing between the cells(FIGS. 5a-5d ).

The D1, D2 or HA16 Media are Capable of Maintaining hESCs with NormalPopulation Doubling—

Similar to cells grown on MEFs, cells cultured with D2 or HA16 medium inall tested culture methods, and the D1 medium in the feeder layer-freesystems, were passaged routinely every four to six days, at the sameratio of 1/2 or 1/3, indicating a similar population doubling time as ofhESCs grown on MEFs. The cells were passage at the same seedingefficiency of about 1 million cells per 10 cm², with the same viabilityrate of over 95%. Using 40% human serum and 10% DMSO, cells weresuccessfully frozen and thawed.

Karyotype Analysis Revealed Normal Karyotype of hESCs Grown with the D1,D2, CM100 or HA16 Media—

15 passages and more after transferring the cells into the testedenvironments, karyotype analysis was performed by Giemsa banding on twoseparate cultures, representing the four medium conditions, D1, D2,CM100 and HA16 at the different culture methods. At least 20 cells weretested from each sample, 40 cells from each medium combination. Allexamined cells were found to sustain normal karyotype of 46,XX for celllines I3 and I4 and 46,XY for cell line I6 (data not shown). Overall,these results suggest that the cells' karyotype remains stable in thetested conditions, similarly to ESCs grown with MEFs using traditionalmethods (Amit et al, 2000).

hESCs Cultured with the D1, D2 or HA16 Express Typical Cell SurfaceMarkers—

Several surface markers typical of primate undifferentiated ES cellswere examined using immunofluorescent staining (Thomson et al, 1995,1996, 1998). hESCs cultured with the D1, D2 or HA16 medium for more than20 passages, while using the tested culture conditions, were found to bestrongly positive to surface markers TRA-1-60 (FIG. 6a ), SSEA4 (FIG. 6b), TRA-1-81 (FIG. 6c ) and Oct 4 (data not shown). As in other primateES cells, staining with SSEA3 was weak and negative for SSEA1 (data notshown).

hESCs Cultured with the D1, D2 or HA16 Medium are Pluripotent as Testedby EBs Formation In Vitro—

The developmental potential of the cells after prolonged culture in thetested culture methods was examined in vitro by the formation ofembryoid bodies (EBs). After more than 15, 20 and 30 passages in mediumD1, D2 and HA16, respectively hESCs formed EBs similar to those createdby hESCs grown on MEFs (not shown). Within these EBs, stem cellsdifferentiated into cell types representative of the three embryonicgerm layers as described for EBs formed from hESCs cultured on otherculture systems (Itskovitz-Eldor et al, 2000).

EBs Formed from the hESCs Cultured on the D1, D2 or HA16 Medium areCapable of Differentiating into the Ectoderm, Endoderm and Mesoderm CellLineages—

While undifferentiated cells cultured in the tested medium, feederlayers and matrices, expressed undifferentiated genetic markers such asOct 4, Nanog, Sox2, Rex1, Cx43 and FGF4 (not shown) [Bhattacharya et al,2004], cells harvested from 10 day-old EBs expressed genes such asalbumin and glucagon (endoderm), α-cardiac actin, β-globulin and Flk1(mesoderm), and AC133 and neurofilament (ectoderm) as demonstrated byRT-PCR analysis (data not shown).

hESCs Cultured with the D1, D2 or HA16 Medium are Pluripotent as Testedby Teratomas Formation In Vivo—

The cells pluripotency was also tested in vivo by teratomas formation.hESCs cultured for over 12 passages in the HA16, D1 or D2 medium, in thetested culture systems formed teratomas following their injection intoSCID-Beige mice. Within these teratomas, hESCs differentiated torepresentative tissues of the three embryonic germ layers including;cartilage, muscle, bone and fat (mesoderm), stratified epithelium,melanin containing epithelium (ectoderm), and kidney like structure(endoderm and mesoderm), and epithelium of endoderm origin (data notshown). Teratomas formation rates of 90%, and the number of injectedcells were identical to those demonstrated by cells cultured usingtraditional methods (Amit et al, 2000).

Altogether, these results demonstrate that hESCs cells subjected toprolonged culture in the tested culture systems demonstrated all hESCsfeatures including; pluripotency, chromosomal stability, expression ofspecific genes and surface markers and indefinite proliferation asundifferentiated cells.

Example 3 Evaluation of the Capacity of the TGF β-Containing MediaDevoid of Serum, Serum Replacement and Albumin to Support Derivation ofhESC Line on Foreskin Fibroblasts in a Complete Xeno-Free System

Materials and Experimental Methods—

as in Example 2, hereinabove.

Experimental Results

The HA16 and D2 Media are Suitable for Derivation of hESC Line onForeskin Fibroblast Feeder Layers in a Complete Xeno-Free System—

The medium combinations of the present invention were also tested forthe ability to support hESC line derivation. While using the HA16 or D2medium on foreskin fibroblasts as a supportive layer, new hESC lineswere successfully derived and maintained for at least 2 passages (in thepresence of the D2 medium) or at least 18 passages (in the presence ofthe HA16 medium). The hESC line derived on foreskin in the presence ofthe HA16 culture medium demonstrated stem cells morphology at passage 18(and the culture is still ongoing), normal XY karyotype and pluripotencyas evidenced by the formation of EBs (FIGS. 3a-3b and data not shown).The growth and success rates were similar to those obtained while usingtraditional culture methods. Since the used foreskin fibroblasts line,F21, were derived without any animal products, this new hESC lines werederived under complete xeno-free conditions. Although the new hESC linesstill need to be tested for additional hESCs features, their morphologyand proliferation rates indicate a typical hESCs culture.

Example 4 TGF β-Containing Medium Devoid of Serum, Serum Replacement andAlbumin is Suitable for Expanding and Maintaining hESCs in Suspension

To examine the possibility of using the TGFβ-containing media devoid ofserum, serum replacement and protein carrier (albumin) for expanding andmaintaining hESCs in an undifferentiated state, hESCs were cultured insuspension, as follows.

Materials and Experimental Methods

ESCs and Culture Media—

ESC cultures and the tested media: D1 medium, D2 medium, HA16 medium,HA19 medium and HACM100 medium, which do not contain serum or serumreplacement; and the CM100F medium which contains serum replacement,were as described in Example 2, hereinabove.

Culture in Suspension—

To examine the possibility of using the TGFβ-containing medium which isdevoid of serum, serum replacement and albumin for scalable culture ofhESCs in suspension, hESCs were cultured in suspension in 58 mm petridishes (Greiner, Frickenhausen, Germany) in a cell density of 1.5×10⁶ to6×10⁶. The HA16 medium was supplemented with 0.1% F68 (Sigma, St. Louis,Mo., USA) for the suspended culture. The cells were passage every 5-7days using either 30-60 minute incubation with 1.5 mg/ml type IVCollagenase (Worthington biochemical corporation, Lakewood, N.J., USA)or 25 minutes incubation with 1.5 mg/ml type IV Collagenase followed byfive minutes incubation with 1 mg/ml Dispase (Invitrogen Corporationproducts, Grand Island N.Y., USA), and further broken into small clumpsusing 200 μl Gilson pipette tips. Alternatively, the cells were passagedmechanically using 27 g needles. The medium was changed on a dailybasis. Following continuous culturing under these conditions the cellswere tested for hESC characteristics. The basic media used for culturinghESCs in suspension (which can be further supplemented with the additiveand growth factors as described hereinabove) were DMEM, ko-DMEM,DMEM/F12, MabADCB or NCTC medium.

Derivation of New hESC Lines in a Suspension Culture with the TGFβ-Containing Medium Devoid of Serum, Serum Replacement and Albumin

Blastocyst Cultivation—

Zygotes were donated by couples undergoing PGD or in vitro fertilization(IVF) at Cornell Medical College, N.Y., who signed informed consentforms. The couples underwent the traditional IVF procedure after ovarianstimulation with gonadotropins and oocyte retrieval. Zygotes werecultured to the blastocyst stage according to IVF laboratory standardprotocol: under oil using specialized C1/C2 media for insemination,growth and blastocyst development (Cornell).

Derivation of hESC Lines in a Suspension Culture—

Following the removal of the zona pellucida using Tyrode's acidicsolution (Sigma, St Louis Mo., USA), the trophoblast layer isspecifically removed either by immunosurgery or mechanically using 27 gneedles. The exposed ICM is further cultured in suspension culture witha suitable culture medium (e.g., the CM100F, HA16 or D2) for 4-10 days.Initially, the cells are mechanically split using 27 g needles.

RT PCR Analysis—

Total RNA was isolated from hESCs grown for 10-15 passages in thesuspension culture using Tri-Reagent (Sigma, St. Louis Mo., USA),according to the manufacturer's instructions. cDNA was synthesized from1 μg total RNA using MMLV reverse transcriptase RNase H minus (Promega,Madison Wis., USA). PCR reactions included denaturation for 5 minutes at94° C. followed by repeated cycles of 94° C. for 30 seconds, annealingfor 30 seconds at an annealing temperature as specified in Table 1,hereinabove and extension at 72° C. for 30 seconds. PCR primers andreaction conditions used are described in Table 1, hereinbelow. PCRproducts were size-fractionated using 2% agarose gel electrophoresis.DNA markers were used to confirm the size of the resultant fragments.

EB Formation from hESCs Cultured in Suspension—

For the formation of EBs, one to three 58 mm petri dishes (Greiner,Frickenhausen, Germany) containing ESCs in suspension cultures weretransferred to new 58 mm petri dishes containing EBs-differentiationmedium consisting of 80% DMEM/F12 (Biological Industries, Beit Haemek,Israel), supplemented with 20% FBSd (HyClone, Utah, USA), and 1 mML-glutamine (Invitrogen Corporation, Grand Island N.Y., USA).Alternatively, prior to their transfer to the EB-differentiation medium,the ESCs were subject to treatment with 1 mg/ml type IV collagenase andfurther broken into small clumps using 1000 μl Gilson pipette tips. 10day-old EBs were harvested for RNA isolation and histologicalexamination.

Immunohistochemistry, Karyotype Analysis and Teratoma Formation—

were performed as described in Example 2, hereinabove.

Experimental Results

The CM100F, HA16, D1, D2 and HA19 Media are Suitable for Culturing hESCsin Suspension—

hESCs were cultured in suspension using the newly developedTGFβ-containing medium types which are devoid of serum, serumreplacement and albumin. To date, the highest passage of hESCs grown insuspension in the tested medium types were 3 passages in the D1 medium,7 passages in the D2 medium, 10 passages in the HA19 medium and 17passages in the CM100F medium. All hESCs exhibited undifferentiatedmorphology at these passages and can be further cultured in these mediaand maintain hESCs features. Histological sections of the hESCs clumpsformed in the suspension cultures illustrated homogeneous cellpopulation, of round cells with large nucleus (FIGS. 9a-9g ). Inaddition, when the cells were plated back on MEFs, they created colonieswith typical hESCs morphology (FIGS. 9b-9e ), and if returned tosuspension cultures, they continued proliferation as undifferentiatedcells (data not shown). When hESCs were cultured in a suspension culturein the presence of the serum or serum replacement-free,IL6RIL6-containing HACM100 medium, the cells were expanded andmaintained in the undifferentiated state for at least 1-2 passages (datanot shown).

hESCs Cultured in Suspension in the Presence of the D1, D2, HA19 orCM100F Media Express Markers of Undifferentiated hESCs—

Cells cultured in suspension in the presence of the D2 medium for 3passages as small clumps of 200-1500 cells expressed stem cells markerssuch as Oct 4 (FIG. 8a ), TRA-1-60 (FIG. 8b ), TRA-1-81 (FIG. 8c ) andSSEA4 (data not shown). Similar results were obtained with the CM100F,D1 or D2 medium at passage 5 (p-5) (data not shown). When cultured insuspension culture in the presence of the CM100F or the HA19 medium thecells expressed high levels of typical stem cells markers such as Oct 4(FIG. 10a ), Rex1 (FIG. 10b ), Sox2 (FIG. 10c ), Nanog (FIG. 10d ) andFGF4 (data not shown) as demonstrated by RT-PCR analysis.

ESCs Cultured in Suspension are Capable of Forming EBs—

When removed from the D1, D2 or HA16 medium and transferred to EBsmedium (80% DMEM/F12 supplemented with 20% FBSd and 1 mM L-glutamine),the cells formed EBs containing representative tissues of threeembryonic germ layers.

Rhesus ESCs can be Also Cultured in the Suspension Cultures of thePresent Invention—

Similar results with Rhesus ESCs (monkey embryonic stem cells, lineR366.4, University of Wisconsin, primate center, Thomson lab, Madison,Wis.), which are regarded as good candidate for transgenic model tohuman diseases, were obtained when the Rhesus ESCs were cultured insuspension in the HA16, D1 and D2 TGFβ-containing culture media (datanot shown).

Thus the new TGFβ-containing medium, which is devoid of serum, serumreplacement and albumin, or the IL6RIL6-containing medium are capable ofsupporting the undifferentiated culture of hESCs, while maintaininghESCs characteristics, and provide methods for massive culture of thesecells for industrial and clinical purposes.

Analysis and Discussion

hESCs, like mouse ES cells, are traditionally cultured with MEFs, whichmay expose them to animal pathogens. In this study, the presentinventors have demonstrated, for the first time, a defined animal, serumand feeder layer-free culture system for hESCs, based on the use ofcommercial medium supplemented with either TGFβ₃ or TGFβ₁ and bFGF, andhuman fibronectin matrix as substitute. This medium is designed formassive cultivation of cells in GMP for industrial or clinical purposes.All medium types of the present invention (with TGFβ₃ or TGFβ₁), supporthESCs culture. The culture medium with the TGFβ isoform 3 was superiorof the culture medium with the TGFβ1 isoform in terms of less backgrounddifferentiation. All medium types of the present invention support theculture with feeders as good as with the regular serum containing media.Cells retained the same proliferation rates and the same backgrounddifferentiation percentages as hESCs cultured with MEFs usingtraditional culture methods. Furthermore, the medium can also be usedfor massive suspended culture of undifferentiated hESCs.

Two isoforms of TGFβ, TGFβ₃ and TGFβ₁, were tested for their ability tomaintain hESCs in an undifferentiated state using various cultureconditions. TGFβ₃ (D2 and HA16 media) was found to be the most suitablemedium supplement, supporting undifferentiated culture of hESCs whileusing all the tested culture possibilities. All hESCs, from threedifferent cell lines, continued to proliferate while retaining normalhESC features throughout the prolonged culture. Medium supplemented withTGFβ₁ (D1 medium) on the contrary, was demonstrated to supportundifferentiated hESC culture only while using feeder layer free culturesystems.

Cells cultured while using these media (D1, D2, and HA16) maintained allthe characteristics of ESCs. After prolonged culture of more than 20passages, the cells remained undifferentiated, as demonstrated by thecolony and single cell morphology, and by the expression of markerstypical of undifferentiated primate ESCs [Thomson et al, 1995, 1996,1998; Reubinoff et al, 2000]. In addition, while cultured in theseconditions, hESCs expressed specific markers for the undifferentiatedstage such as Oct 4, Sox 2, Rex1 and Nanog, as demonstrated by RT-PCR.

Karyotype analysis carried out on representative cell samplesdemonstrated that the hESCs' karyotype remained stable in the proposedconditions. None of the examined cells exhibited any karyotypeabnormalities.

The cells' pluripotency was examined in vitro. Cells cultured in thetested culture systems for more than 10 passages, formed EBs similar tothose created when grown on MEFs [Itskovitz-Eldor et al, 2000]. RT-PCRanalysis demonstrated that cells within these EBs differentiated intodifferent cell types representative of the three germ layers.Furthermore, following their injection to SCID-Beige mice, hESCs formedteratomas containing a multitude of tissues types (D1 and D2, HA16 inprocess). The teratoma formation rates were identical to those of cellscultured with MEFs. Thus, the pluripotency of the cells culturecontinuously in the tested culture methods remained intact.

Additionally, and most importantly, the same measurements were used tocharacterize cells cultured with the D1, D2 and HA16 medium insuspension. ESCs cultured under these conditions for more than 7passages exhibited undifferentiated markers and when transferred todifferentiation promoting conditions, demonstrated pluripotency. Thus,these media can enable massive culture of undifferentiated hESCs, andfacilitate the development of control bioprocesses in industrialbioreactors.

These results demonstrate that hESCs can be maintained asundifferentiated cells in the proposed defined animal- and serum-freemedium combination, without any feeder cells (D1, D2 and HA16) oralternatively, with commonly used acceptable feeder layers (D2 andHA16). Thus, these media can facilitate hESCs culture for research,industrial and clinical purposes. Moreover, these novel culture mediawere found to support suspended culture of undifferentiated hESCs, thefirst and primary step in developing a massive culture system for theirgrowth and scale-up, a crucial step for any industrial and clinicaluses.

The mechanism by which hESCs self-maintain is still unclear.Accumulating data suggest the involvement of TGFβ family members inhESCs renewal [Amit et al, 2004; Ludwig et al, 2006; James et al, 2005;Chen et al, 2006, Valdimarsdottir & Mummery, 2006]. Furthercomplementary research is required to explain the underlying mechanismsof action of TGFβ at the level of signal transduction, and the fact thatTGFβ₃ is more potent than TGFβ₁.

Future clinical uses of hESCs will require a reproducible, well-definedand xeno-free culture system. The culture method described in this studyof fibronectin and D1, D2 or HA16 medium and foreskins fibroblast meetthese needs. The well-defined media demonstrated in the present studyare suitable for culturing hESCs and may be advantageous for undertakingresearch on the mechanisms of ESC self-maintenance, especially of thepossible roles of the TGFβ pathway. Other studies using hESCs, such asthe research on differentiation pathways and mechanisms, will benefitfrom the availability of a well-defined and reproducible culture system.

Thus, the present invention discloses for the first time:

1. A defined, xeno-free, serum, serum replacement or albumin-free systemsuitable for both culture and derivation of hESCs.

2. Defined medium combinations, highly effective in supporting hESCsculture in variety of culture conditions. Priority of TGFβ₃ over TGFβ₁.TGFβ₃ was never demonstrated to promote self-renewal of stem cells.

3. A culture system that allows hESC culturing in suspension asundifferentiated without a carrier (without substrate adherence).

4. A scalable culture system, suitable for developing controlbioprocesses in industrial bioreactors.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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What is claimed is:
 1. A method of culturing primate pluripotent stemcells in a pluripotent and undifferentiated state, the method comprisingculturing the primate pluripotent stem cells in a suspension culturewhich comprises a culture medium, said culture medium comprising basicfibroblast growth factor (bFGF) and at least one factor selected fromthe group consisting of transforming growth factor beta-3 (TGFβ3)isoform, transforming growth factor beta-1 (TGFβ1) isoform, and IL6RIL6chimera, wherein the primate pluripotent stem cells are expanded for atleast 5 passages in said suspension culture without adherence to anexternal substrate, wherein the primate pluripotent stem cells aremaintained in said suspension culture in a pluripotent andundifferentiated state, wherein the pluripotent stem cells express OCT4,and are capable of forming teratomas containing cells of the mesoderm,ectoderm and endoderm embryonic germ layers.
 2. The method of claim 1,wherein said culturing is in a bioreactor.
 3. The method of claim 1,wherein the primate pluripotent stem cells comprise primate embryonicstem cells.
 4. The method of claim 1, wherein said culture medium isdevoid of serum.
 5. The method of claim 1, wherein said culture mediumis devoid of serum replacement.
 6. The method of claim 1, wherein saidculture medium is animal contaminant-free.
 7. The method of claim 1,wherein said suspension culture is feeder cell layer-free.
 8. The methodof claim 1, wherein said culture medium is protein carrier-free.
 9. Themethod of claim 1, wherein said IL6RIL6 chimera comprises amino acids112-355 of SEQ ID NO:
 32. 10. The method of claim 1, wherein saidIL6RIL6 chimera is provided at a concentration of at least 25 ng/ml. 11.The method of claim 1, wherein said bFGF is provided at a concentrationof at least 2 ng/ml.
 12. The method of claim 1, wherein said TGFβ3 is ata concentration of at least 0.5 ng/ml.
 13. The method of claim 1,wherein said TGFβ1 is at a concentration of at least 0.06 ng/ml.
 14. Themethod of claim 1, wherein said primate pluripotent stem cells are humanpluripotent stem cells.
 15. The method of claim 1, wherein said primatepluripotent stem cells are monkey pluripotent stem cells.